Immunogenic composition and vaccine containing African swine fever virus peptides and proteins and uses thereof

Abstract

The present invention relates to African swine fever virus (ASFV) peptides and / or polypeptides and immunogenic fragments thereof, corresponding AFSV-encoding oligonucleotides and / or polynucleotides and immunogenic fragments thereof, immunogenic compositions, vaccines and uses thereof.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that complies with 37 CFR 1.821-1.825. The sequence listing accompanying this application is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of veterinary vaccines, and specifically to African swine fever virus peptides and / or polypeptides, preferably full-length proteins and immunogenic fragments thereof, corresponding oligonucleotides and / or polynucleotides encoding African swine fever virus and immunogenic fragments thereof, immunogenic compositions, vaccines and their uses. Background Art

[0004] African swine fever (ASF) was introduced to Georgia in 2007 and subsequently spread throughout continental Europe after spreading in Asia since 2018, proving to be a major threat to the global pig industry. ASF is a hemorrhagic disease of pigs with a mandatory declaration from the World Organization for Animal Health (OIE) and causes significant economic losses to affected countries. The causative agent, African swine fever virus (ASFV), is a large, enveloped icosahedral virus with a dsDNA genome of approximately 180 kbp in length. Currently, there is no commercial vaccine against ASFV. Early and effective diagnosis, followed by slaughter of infected and contact animals, is the only control method currently recommended by the OIE, a measure that is unfortunately unaffordable in vulnerable regions.

[0005] ASF vaccine development is largely hampered by a lack of knowledge about key aspects of ASFV infection and protective immunity. In this regard, CD8+ T lymphocytes have been widely shown to play a crucial role in protective responses against ASFV. However, the identities of ASFV antigens capable of inducing protective CD8+ T cell responses remain largely unknown. Identification of these protective antigens could lead to fundamental vaccine design principles and a better understanding of the mechanisms underlying ASFV immunity.

[0006] The feasibility of providing protection against the Georgia 2007 / 1 isolate using live attenuated virus has been demonstrated (Monteagudo et al., 2017; O'Donnell et al., 2015), demonstrating the presence of protective Georgia 2007 / 1 antigens. However, while DNA vaccination has been shown to successfully identify antigens or epitopes with protective potential against E75 ASFV (Argilaguet et al., 2012; Lacasta et al., 2014), these results were not reproducible against the highly virulent Georgian isolate. Other DNA-based vaccine formulations have demonstrated ASFV-specific responses but have again failed to confer protection against the Georgia 2007 / 1 isolate / strain (Jancovich et al., 2018; Lopera-Madrid et al., 2017).

[0007] Another prior art is as follows:

[0008] Farlow J et al. (Virology Journal 2018, 15(1):190) describe intra-epidemic genomic variation in the highly pathogenic African swine fever virus (ASFV) from Georgia.

[0009] The Uniprot database revealed the ASFV Georgia 2007 / 1 full CDS protein A118R with accession number A0A2X0RVA9.

[0010] De Villiers EP et al. (Virology 2010, 400: 128-136) describe a phylogenetic analysis of 11 complete African swine fever virus genome sequences.

[0011] Netherton CL et al. (Front Immunol 2019, 10: 1318) describe the recognition and immunogenicity of African swine fever virus antigens.

[0012] Sánchez EG et al. (Virus Research 2019, 265: 150-155) describe the development of a vaccine against African swine fever virus.

[0013] WO 2017 / 096341 discloses a multivalent vaccine using adenovirus as a vector.

[0014] There remains an unmet need for a safe ASF vaccine that specifically confers protection against the Georgia 2007 / 1 ASFV isolate / strain. Summary of the Invention

[0015] To overcome the deficiencies in the prior art, the present invention provides an immunogenic composition comprising:

[0016] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0017] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0018] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and

[0019] (d) optionally one or more pharmaceutically or veterinarily acceptable carriers or excipients, preferably suitable for oral, intradermal, intramuscular or intranasal administration.

[0020] The present invention further relates to a vaccine or pharmaceutical composition comprising:

[0021] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0022] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0023] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and

[0024] (d) one or more pharmaceutically or veterinarily acceptable carriers or excipients, preferably suitable for oral, intradermal, intramuscular or intranasal administration;

[0025] The vaccine or pharmaceutical composition optionally further contains an adjuvant.

[0026] The present invention further relates to an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein for use in a method for reducing or preventing clinical symptoms or diseases caused by infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, or for use in a method for treating and / or preventing infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, wherein the at least one African swine fever virus, preferably a pathogenic African swine fever virus, is infected with The clinical symptoms or diseases caused by infection or the at least one African swine fever virus, preferably the pathogenic African swine fever virus infection is preferably selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia. The present invention is also intended to include corresponding methods for reducing or preventing clinical symptoms or diseases caused by infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, or corresponding methods for treating and / or preventing infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus (which comprises administering to the porcine animals, preferably pigs, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein), as well as corresponding uses of the immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for reducing or preventing clinical symptoms or diseases caused by infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, or for treating and / or preventing infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus.

[0027] The present invention further relates to an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for use in a method for immunizing a porcine animal, preferably a pig, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a porcine animal, preferably a pig, the method comprising the steps of: administering an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein to a porcine animal, preferably a pig, wherein the immunogenic composition or vaccine or pharmaceutical composition does not cause clinical symptoms of infection but is capable of inducing the porcine animal, preferably a pig, to resist at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a method for immunizing a porcine animal, preferably a pig, to resist at least one African swine fever virus, preferably a pathogenic African swine fever virus, the method comprising the steps of: administering an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein to a porcine animal, preferably a pig, wherein the immunogenic composition or vaccine or pharmaceutical composition does not cause clinical symptoms of infection but is capable of inducing the porcine animal, preferably a pig, to resist at least one African swine fever virus, preferably a pathogenic African swine fever virus, It is an immune response of pigs to the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus, wherein the clinical disease or the clinical infection symptoms are preferably selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia. The present invention is also intended to include a corresponding method for immunizing a porcine animal, preferably a pig, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a porcine animal, preferably a pig, comprising the steps of administering to a porcine animal, preferably a pig, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein, wherein the immunogenic composition or vaccine or pharmaceutical composition does not cause clinical symptoms of infection but is capable of inducing immunity of the porcine animal, preferably a pig, to the at least one African swine fever virus, preferably the pathogenicity of the at least one African swine fever virus. and the corresponding use of an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for immunizing porcine animals, preferably pigs, against clinical disease caused by at least one African swine fever virus, preferably pathogenic African swine fever virus, in porcine animals, preferably pigs, wherein the immunogenic composition, vaccine or pharmaceutical composition does not cause clinical symptoms of infection but can induce an immune response that immunizes porcine animals, preferably pigs, against the at least one African swine fever virus, preferably the at least one pathogenic form of African swine fever virus.

[0028] The present invention further relates to an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for use in a method for prime-boost immunization of porcine animals, preferably pigs, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus in porcine animals, preferably pigs, the method comprising the following steps: administering to porcine animals, preferably pigs, once or twice an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein containing: (i) as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof; or (ii) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, which contains as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof (priming step) ); and subsequently - after one or two primings - administering a live attenuated African swine fever virus, preferably BA71ΔCD2, to a porcine animal, preferably a pig (boosting step); wherein the immunogenic composition or vaccine or pharmaceutical composition and the live attenuated African swine fever virus independently of each other do not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal, preferably the pig, against the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus, wherein the clinical disease or the clinical infection symptoms are preferably selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin mottling, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleeds, abortion, leukopenia, thrombocytopenia.The present invention is also intended to include a method for prime-boost immunization of porcine animals, preferably pigs, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in porcine animals, preferably pigs, the method comprising the following steps: administering to porcine animals, preferably pigs, once or twice an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein containing: (i) as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof; or (ii) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, which contains as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof and subsequently - after one or two primings - administering a live attenuated African swine fever virus, preferably BA71ΔCD2, to a porcine animal, preferably a pig (boosting step); wherein the immunogenic composition or vaccine or pharmaceutical composition and the live attenuated African swine fever virus independently of each other do not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal, preferably the pig, against the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus; and the corresponding use of the immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for prime-boost immunization of porcine animals, preferably pigs, against clinical diseases caused by at least one African swine fever virus, preferably pathogenic African swine fever virus in porcine animals, preferably pigs.

[0029] The present invention further relates to a kit for vaccinating porcine animals, preferably pigs, against diseases associated with at least one African swine fever virus (preferably a pathogenic African swine fever virus) in porcine animals, preferably pigs, and / or reducing the incidence or severity of one or more clinical symptoms associated with at least one African swine fever virus (preferably a pathogenic African swine fever virus) or caused by at least one African swine fever virus (preferably a pathogenic African swine fever virus), the kit comprising:

[0030] (a) a dispenser capable of administering the vaccine to the porcine animal; and

[0031] (b) an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein; and

[0032] (c) optional instruction booklet;

[0033] The disease or clinical symptoms are preferably selected from the group consisting of African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival secretions, bleeding, nosebleed, abortion, leukopenia, and thrombocytopenia.

[0034] The present invention further relates to African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising, preferably consisting of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,19,21,23,25,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,4 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 8 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 8, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、272、274、275、276、277、278、279、280、281、282、283、284、285、287、289、290、291、292、293、294、295、297、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、420、422、423、424、425、426、427、428、429、430、432、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、458、460、461、462、463、464、465、466、468、470、471、472、473、474、475、476、477、478、479、481、483、484、485、486、487、489、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、518、520、521、522、523、524、526、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、568、570、572、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、719、721、722、724、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、774、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854。、

[0035] The present invention further relates to African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising, preferably consisting of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 569, 571, 572, 573, 574, 575), D1134L (SEQ ID NO: 569, 571, 574, 575), D1135L (SEQ ID NO: 569, 571, 574, 575), D1136L (SEQ ID NO: 569, 571, 574, 575), D1137L (SEQ ID NO: 569, 571, 574, 575), D1138L (SEQ ID NO: 569, 571, 574, 575), D1139L (SEQ ID NO: 569, NO:297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570), K145R (SEQ ID NO:526, 524, 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO: 468, 466, 465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 489, 487, 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO: 432, 430, 429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ ID NO: 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ IDNO:733, 732, 731, 730, 729, 728, 727, 726), P1192R (SEQ IDNO: 817, 816, 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 692, 691, 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO: 703, 702, 701, 700, 699), EP424R (SEQ ID NO:389, 388, 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO: 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 75, 74, 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 278, 277, 276, 275), D339L (SEQ ID NO: 322, 321, 320), D117L (SEQ ID NO: 862, 864), I243L (SEQ ID NO: 494, 493, 492, 491), I73R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 513), MGF505-7R / MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO:NO:568、566、565、564、563)、D1133L(SEQ ID NO:297、295、294、293、292、291、290、289、287、285、284、283、282、281)、CP312R(SEQ ID NO:274、272、269、268、267)、A240L(SEQID NO:854、853、25)。

[0036] The present invention further relates to African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, wherein the African swine fever virus polypeptide, preferably the full-length protein, comprises the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: A238L (SEQ ID NO: 21), A238L (SEQ ID NO: 23), A240L (SEQ ID NO: 853), A240L (SEQ ID NO: 854), B475L (SEQ ID NO: 65), B475L (SEQ ID NO: 66), CP2475 (SEQ ID NO: 256), CP2475 (SEQ ID NO: 257), CP312R (SEQ ID NO: 272), CP312R (SEQ ID NO: 273), NO:274), D1133L (SEQ ID NO:295), D1133L (SEQ ID NO:297), EP402R (SEQ ID NO:378), EP424R (SEQ ID NO:388), EP424R (SEQ ID NO:389), G1211R (SEQ ID NO:430), G1211R (SEQ ID NO:432), H339R (SEQ ID NO:466), H339R (SEQ ID NO:468), I226R (SEQ ID NO:487), I226R (SEQ ID NO:489), K145R (SEQ ID NO:524), K145R (SEQ ID NO:526), ​​M448R (SEQ ID NO:566), M448R (SEQ ID NO:568), M1249L (SEQ ID NO:561), M1249L (SEQ ID NO:562), MGF_100-1L / MGF100-1L (SEQ ID NO:572), MGF505-1R / MGF_505-1R (SEQ ID NO:691), MGF505-1R / MGF_505-1R (SEQ ID NO:692), MGF505-8R / MGF_505-8R (SEQ ID NO:722), MGF505-7R / MGF_505-7R (SEQ ID NO:724), MGF505-8R / MGF_505-8R (SEQ ID NO:772), MGF505-7R / MGF_505-7R (SEQ IDNO: 774), MGF505-9R / MGF_505-9R (SEQ ID NO: 732), MGF505-9R / MGF_505-9R (SEQ ID NO: 733), P1192R (SEQ ID NO: 816), P1192R (SEQ ID NO: 817); and / or wherein the African swine fever virus peptide and / or its immunogenic fragment and / or African swine fever virus polypeptide / full-length protein immunogenic fragment contains the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of A238L (SEQ ID NO: 17), A238L (SEQ ID NO: 19), A240L (SEQ ID NO: 25), B475L (SEQ ID NO: 26), NO:48), B475L (SEQ ID NO:49), B475L (SEQ ID NO:50), B475L (SEQ ID NO:51), B475L (SEQ ID NO:52), B475L (SEQ ID NO:53), B475L (SEQ ID NO:54), B475L (SEQ ID NO:55), B475L (SEQ ID NO:56), B475L (SEQ ID NO:57), B475L (SEQ ID NO:58), B475L (SEQ ID NO:59), B475L (SEQ ID NO:60), B475L (SEQ ID NO:61), B475L (SEQ ID NO:62), B475L (SEQ ID NO:63), B475L (SEQ ID NO:64), CP2475 (SEQ ID NO:235), CP2475 (SEQ ID NO:236), CP2475 (SEQ ID NO:237), CP2475 (SEQ ID NO:238), CP2475 (SEQ ID NO:239), CP2475 (SEQ ID NO:240), CP2475 (SEQ ID NO:241), CP2475 (SEQ ID NO:242), CP2475 (SEQ ID NO:243), CP2475 (SEQ ID NO:244), CP2475 (SEQ ID NO:245), CP2475 (SEQ ID NO:246), CP2475 (SEQ ID NO:247), CP2475 (SEQ ID NO:248), CP2475 (SEQ IDNO:249)、CP2475(SEQ ID NO:250)、CP2475(SEQ ID NO:251)、CP2475(SEQ ID NO:252)、CP2475(SEQID NO:253)、CP2475(SEQ ID NO:254)、CP2475(SEQ ID NO:255)、CP2475L(p37)(SEQ IDNO:261)、CP2475L(p37)(SEQ ID NO:262)、CP2475L(p37)(SEQ ID NO:263)、CP2475L(p37)(SEQ ID NO:264)、CP2475L(p37)(SEQ ID NO:265)、CP2475L(p37)(SEQ ID NO:266)、CP2475L(p150)(SEQ ID NO:258)、CP2475L(p150)(SEQ IDNO:259)、CP2475L(p150)(SEQ IDNO:260)、CP312R(SEQ ID NO:267)、CP312R(SEQ ID NO:268)、CP312R(SEQ ID NO:269)、D1133L(SEQ ID NO:281)、D1133L(SEQ ID NO:282)、D1133L(SEQ IDNO:283)、D1133L(SEQID NO:284)、D1133L(SEQ ID NO:285)、D1133L(SEQ ID NO:287)、D1133L(SEQ ID NO:289)、D1133L(SEQ ID NO:290)、D1133L(SEQ ID NO:291)、D1133L(SEQ ID NO:292)、D1133L(SEQID NO:293)、D1133L(SEQ ID NO:294)、EP402R(SEQ ID NO:372)、EP402R(SEQ IDNO:373)、EP402R(SEQ ID NO:374)、EP402R(SEQ ID NO:375)、EP402R(SEQ ID NO:376)、EP402R(SEQID NO:377)、EP424R(SEQ ID NO:379)、EP424R(SEQ ID NO:380)、EP424R(SEQ ID NO:381)、EP424R(SEQ ID NO:382)、EP424R(SEQ ID NO:383)、EP424R(SEQ ID NO:384)、EP424R(SEQIDNO:385)、EP424R(SEQ ID NO:386)、EP424R(SEQ ID NO:387)、G1211R(SEQ ID NO:416)、G1211R(SEQ ID NO:417)、G1211R(SEQ ID NO:418)、G1211R(SEQ ID NO:420)、G1211R(SEQID NO:422)、G1211R(SEQ ID NO:423)、G1211R(SEQ ID NO:424)、G1211R(SEQ ID NO:425)、G1211R(SEQ ID NO:426)、G1211R(SEQ ID NO:427)、G1211R(SEQ ID NO:428)、G1211R(SEQID NO:429)、H339R(SEQ ID NO:454)、H339R(SEQ ID NO:455)、H339R(SEQ ID NO:456)、H339R(SEQ ID NO:458)、H339R(SEQ ID NO:460)、H339R(SEQ ID NO:461)、H339R(SEQ IDNO:462)、H339R(SEQ ID NO:463)、H339R(SEQ ID NO:464)、H339R(SEQ ID NO:465)、I226R(SEQ IDNO:478)、I226R(SEQ ID NO:479)、I226R(SEQ ID NO:481)、I226R(SEQ ID NO:483)、I226R(SEQID NO:484)、I226R(SEQ ID NO:485)、I226R(SEQ ID NO:486)、K145R(SEQID NO:514)、K145R(SEQ ID NO:515)、K145R(SEQ ID NO:516)、K145R(SEQ ID NO:518)、K145R(SEQ ID NO:520)、K145R(SEQ ID NO:521)、K145R(SEQ ID NO:522)、K145R(SEQ IDNO:523)、M448R(SEQ ID NO:563)、M448R(SEQ ID NO:564)、M448R(SEQ ID NO:565)、M1249L(SEQ ID NO:539)、M1249L(SEQID NO:540)、M1249L(SEQ ID NO:541)、M1249L(SEQ ID NO:542)、M1249L(SEQ IDNO:543)、M1249L(SEQ ID NO:544)、M1249L(SEQ ID NO:545)、M1249L(SEQ ID NO:546)、M1249L(SEQ IDNO:547)、M1249L(SEQ ID NO:548)、M1249L(SEQ ID NO:549)、M1249L(SEQ ID NO:550)、M1249L(SEQ ID NO:551)、M1249L(SEQ ID NO:552)、M1249L(SEQ ID NO:553)、M1249L(SEQ IDNO:554)、M1249L(SEQ ID NO:555)、M1249L(SEQ ID NO:556)、M1249L(SEQ ID NO:557)、M1249L(SEQ ID NO:558)、M1249L(SEQ ID NO:559)、M1249L(SEQ ID NO:560)、MGF_100-1L / MGF100-1L(SEQ ID NO:570)、MGF505-1R / MGF_505-1R(SEQID NO:684)、MGF505-1R / MGF_505-1R(SEQ ID NO:685)、MGF505-1R / MGF_505-1R(SEQ IDNO:686)、MGF505-1R / MGF_505-1R(SEQ ID NO:687)、MGF505-1R / MGF_505-1R(SEQ ID NO:688)、MGF505-1R / MGF_505-1R(SEQ ID NO:689)、MGF505-1R / MGF_505-1R(SEQ ID NO:690)、MGF505-8R / MGF_505-8R(SEQ ID NO:717)、MGF505-7R / MGF_505-7R(SEQ ID NO:719)、MGF505-7R / MGF_505-7R(SEQ ID NO:721)、MGF505-9R / MGF_505-9R(SEQ ID NO:726)、MGF505-9R / MGF_505-9R(SEQ ID NO:727)、MGF505-9R / MGF_505-9R(SEQ ID NO:728)、MGF505-9R / MGF_505-9R(SEQ ID NO:729)、MGF505-9R / MGF_505-9R(SEQ ID NO:730)、MGF505-9R / MGF_505-9R(SEQ ID NO:731)、P1192R(SEQID NO:801)、P1192R(SEQ ID NO:802)、P1192R(SEQ ID NO:803)、P1192R(SEQ ID NO:804)、P1192R(SEQ ID NO:805)、P1192R(SEQ ID NO:806)、P1192R(SEQ ID NO:807)、P1192R(SEQ ID NO:808)、P1192R(SEQ ID NO:809)、P1192R(SEQ ID NO:810)、P1192R(SEQ ID NO:811)、P1192R(SEQ ID NO:812)、P1192R(SEQ ID NO:813)、P1192R(SEQ ID NO:814)、P1192R(SEQ ID NO:815)。

[0037] The present invention further relates to African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, wherein the African swine fever virus polypeptide, preferably the full-length protein, comprises the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722), M448R (SEQ ID NO: 568, 566), D1133L (SEQ ID NO: 297, 295), CP312R (SEQ ID NO: 274, 272), A240L (SEQ ID NO: 854, 853), A238L (SEQ ID NO: 856), NO: 23, 21), MGF100-1L (SEQ ID NO: 572), K145R (SEQ ID NO: 526, 524), B475L (SEQ ID NO: 66, 65), H339R (SEQ ID NO: 468, 466), I226R (SEQ ID NO: 489, 487), CP2475 (SEQ ID NO: 489, 487), NO:257), CP2475 (SEQ ID NO:256), G1211R (SEQ ID NO:432, 430), M1249L (SEQ ID NO:562, 561), MGF505-9R (SEQ ID NO:733, 732), P1192R (SEQ ID NO:817, 816), MGF505-1R (SEQ ID NO:692, 691), MGF505-3R (SEQ ID NO:703, 702), EP424R (SEQ ID NO:389, 388), C475L (SEQ ID NO:201, 200), B602L (SEQ ID NO:75, 74), CP530R (SEQ ID NO:278, 277), D339L (SEQ ID NO:322, 321), D117L (SEQ ID NO:862, 864), I243L (SEQ ID NO:494, 493), I73R (SEQ ID NO:504, 503), DP238L (SEQ ID NO:327, 326), I9R (SEQ IDNO: 513, 512); and / or wherein the African swine fever virus peptide and / or immunogenic fragment thereof and / or African swine fever virus polypeptide / full-length protein immunogenic fragment comprises the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 721, 719, 717), M448R (SEQ ID NO: 565, 564, 563), D1133L (SEQ ID NO: 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 513, 512); NO:269, 268, 267), A240L (SEQ ID NO:25), A238L (SEQ ID NO:19, 17), MGF100-1L (SEQ ID NO:570), K145R (SEQ ID NO: 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO:465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO:429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ ID NO: 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:731, 730, 729, 728, 727, 726), P1192R (SEQ IDNO: 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO:701, 700, 699), EP424R (SEQ ID NO: 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO:199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 277, 276, 275), D339L (SEQ ID NO: 320), I243L (SEQ ID NO: 492, 491), I73R (SEQ ID NO: 502), DP238L (SEQ ID NO: 325), I9R (SEQ ID NO: 511, 510).

[0038] The present invention further relates to African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably the full-length protein and / or immunogenic fragments thereof.

[0039] The present invention further relates to African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising the following nucleic acid sequence, preferably consisting of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with a nucleic acid sequence selected from the group consisting of: SEQ ID NO:18, 20, 22, 24, 26, 27, 270, 271, 273, 286, 288, 296, 298, 419, 421, 431, 433, 457, 459, 467 , 469, 480, 482, 488, 490, 517, 519, 525, 527, 567, 569, 571, 573, 718, 720, 723, 725, 773, 775.

[0040] The present invention further relates to African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising the following nucleic acid sequence, preferably consisting of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with a nucleic acid sequence selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 1001), MGF505-7R / MGF505-8R (SEQ ID NO: 1001), MGF505-7R / MGF505-8R (SEQ ID NO: 1001), MGF505-7R / MGF505-8R (SEQ ID NO: 1001), M448R (SEQ ID NO: 1001), NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571), K145R (SEQ ID NO: 527, 525, 519, 517), B475L (SEQ ID NO: 866, 867), H339R (SEQ ID NO: 469, 467, 459, 457), I226R (SEQ ID NO: 490, 488, 482, 480), CP2475L (p37) (SEQ ID NO:868, 869), CP2475L (p150) (SEQ ID NO:870, 871), G1211R (SEQ ID NO:433, 431, 421, 419), M1249L (SEQ ID NO:872, 873), MGF505-9R (SEQ ID NO:874, 875), P1192R (SEQ ID NO:876, 877), MGF505-1R (SEQ ID NO: 878, 879), MGF505-3R (SEQ ID NO: 880, 881), EP424R (SEQ ID NO: 882, 883), C475L (SEQ ID NO: 884, 885), B602L (SEQ ID NO: 886, 887), CP530R (SEQ ID NO: 886, 887), NO:888, 889), D339L (SEQ ID NO:890, 891), D117L (SEQ ID NO:863, 865), I243L (SEQ ID NO:892, 893), I73R (SEQ ID NO:894, 895), DP238L (SEQID NO: 896, 897), I9R (SEQ ID NO: 898, 899); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 859, 298, 296, 288, 286), NO: 573, 571); and most preferably is selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26).

[0041] The present invention further relates to a vector comprising one, two, three or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof as described and / or claimed herein. The vector preferably comprises three African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof, wherein the three African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof are preferably selected from EP402R, CP312R and A240L (multiple epitope-1, ME-1), more preferably comprises the following, most preferably consists of the following: NO:855 consisting of a nucleic acid sequence; or containing thirteen African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof, the thirteen African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof are preferably selected from D1133L, G1211R, M1249L, MGF505-9R, P1192R, CP2475L (p150), B475L, EP424R, H339R, I226R, K145R, MGF505-1R and CP2475L (p37) (multiple epitopes-II, ME-II), more preferably containing the following, most preferably consisting of the following: a nucleic acid sequence selected from the group consisting of SEQ ID NO:856.

[0042] The invention further relates to a host cell, preferably a mammalian host cell, containing a vector as described and / or claimed herein.

[0043] Therefore, the solution to the above technical problem is achieved through the embodiments characterized in this specification and in the scope of the claims, and the present invention in its different aspects is implemented according to the scope of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0045] Figure 1 The following is depicted: IFNγ responses assessed by ELISpot analysis using fibroblasts transfected with the pCMV-Ub-MGF505-7R plasmid as APCs and PBMCs from ASF-convalescent animals as effector cells are shown in black. The number of spots, never exceeding 10, was subtracted from the indicated value when stimulated with fibroblasts transfected with the empty pCMV-Ub plasmid. ASFV-specific IFNγ-SC levels are shown in gray.

[0046] Figure 2 Depicted are IFNγ responses to A238L and MGF100-1L full-length Georgia2007 / 1 proteins assessed by ELISpot analysis using fibroblasts transfected with pCMV-Ub-A238L and pCMV-Ub-MGF100-1L plasmids, respectively, as APCs and PBMCs from ASF-convalescent animals as effector cells.

[0047] Figure 3 Depicted are the percentage survival of pigs primed with selected 15 recombinant plasmids (15 clones) or empty pCMV-Ub plasmid (control) following lethal challenge with Georgia 2007 / 1. Both groups were immunized with a low dose of live attenuated virus (LAV) BA71ΔCD2.

[0048] Figure 4 Depicted are the percentage of surviving pigs in the M448R+MGF505-7R primed (solid line) and control (dashed line) groups following lethal challenge infection with Georgia 2007 / 1.

[0049] Figure 5 Depicted are the percentage of surviving pigs in the multi-epitope primed (solid line) and control (dashed line) groups following lethal challenge with Georgia 2007 / 1. DETAILED DESCRIPTION

[0050] The present invention solves problems inherent in the prior art and provides a unique advance in the current state of the art.

[0051] In general, the present invention relates to immunogenic compositions comprising:

[0052] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0053] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0054] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and

[0055] (d) optionally one or more pharmaceutically or veterinarily acceptable carriers or excipients, preferably suitable for oral, intradermal, intramuscular or intranasal administration;

[0056] Or a vaccine or pharmaceutical composition containing:

[0057] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0058] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[0059] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and

[0060] (d) one or more pharmaceutically or veterinarily acceptable carriers or excipients, preferably suitable for oral, intradermal, intramuscular or intranasal administration;

[0061] (e) The vaccine or pharmaceutical composition optionally further contains an adjuvant.

[0062] In a specific aspect, there is provided an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein, wherein the African swine fever virus is selected from the group consisting of BA71, BA71ΔCD2 and / or Georgia 2007 / 1 virus strains.

[0063] In another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (a), one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, contain, preferably consist of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,19,21,23,25,28,29,30,31,32,33,34,35,36,37,38,39,40,41,4 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 7 6, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 2, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 , 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、272、274、275、276、277、278、279、280、281、282、283、284、285、287、289、290、291、292、293、294、295、297、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、420、422、423、424、425、426、427、428、429、430、432、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、458、460、461、462、463、464、465、466、468、470、471、472、473、474、475、476、477、478、479、481、483、484、485、486、487、489、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、518、520、521、522、523、524、526、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、568、570、572、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、719、721、722、724、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、774、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854。、

[0064] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (a), one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprise, preferably consist of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 569, 571, 572, 573, 574), D1134L (SEQ ID NO: 575, 576, 577, 578), D1135L (SEQ ID NO: 579, 580, 581, 582), D1136L (SEQ ID NO: 583, 584, 585), D1137L (SEQ ID NO: 584, 585 NO:297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570), K145R (SEQ ID NO:526, 524, 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO: 468, 466, 465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 489, 487, 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO: 432, 430, 429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ IDNO: 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:733, 732, 731, 730, 729, 728, 727, 726), P1192R (SEQ ID NO:817, 816, 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 692, 691, 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO: 703, 702, 701, 700, 699), EP424R (SEQ ID NO:389, 388, 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO: 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 75, 74, 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 278, 277, 276, 275), D339L (SEQ ID NO: 322, 321, 320), D117L (SEQ ID NO: 862, 864), I243L (SEQ ID NO: 494, 493, 492, 491), I73R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ IDNO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25).

[0065] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (b), one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encode one, two or more African swine fever virus peptides and / or polypeptides as disclosed and / or claimed herein, preferably the full-length protein and / or immunogenic fragment thereof.

[0066] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (b), one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encode one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising, preferably consisting of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 719, 721), M448R (SEQ ID NO: 719, 72 ... NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570), K145R (SEQ ID NO:526, 524, 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO: 468, 466, 465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 489, 487, 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO: 432, 430, 429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ IDNO: 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:733, 732, 731, 730, 729, 728, 727, 726), P1192R (SEQ ID NO:817, 816, 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 692, 691, 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO: 703, 702, 701, 700, 699), EP424R (SEQ ID NO:389, 388, 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO: 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 75, 74, 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 278, 277, 276, 275), D339L (SEQ ID NO: 322, 321, 320), D117L (SEQ ID NO: 862, 864), I243L (SEQ ID NO: 494, 493, 492, 491), I73R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ IDNO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25).

[0067] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (b), one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, contain the following nucleic acid sequence, preferably consist of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO:18, 20, 22, 24, 26, 27, 270, 271, 273, 286, 288, 296, 298, 419, 421, 431, 433, 457, 459, 467 , 469, 480, 482, 488, 490, 517, 519, 525, 527, 567, 569, 571, 573, 718, 720, 723, 725, 773, 775.

[0068] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (b), one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, contain the following nucleic acid sequence, preferably consist of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to a nucleic acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 5 NO:859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571), K145R (SEQ ID NO: 527, 525, 519, 517), B475L (SEQ ID NO: 866, 867), H339R (SEQ ID NO: 469, 467, 459, 457), I226R (SEQ ID NO: 469, 467, 459, 457) NO:490, 488, 482, 480), CP2475L (p37) (SEQ ID NO: 868, 869), CP2475L (p150) (SEQ ID NO: 870, 871), G1211R (SEQ ID NO: 433, 431, 421, 419), M1249L (SEQ ID NO: 872, 873), MGF505-9R (SEQ ID NO: 874, 875), P1192R (SEQ ID NO: 876, 877), MGF505-1R (SEQ ID NO: 878, 879), MGF505-3R (SEQ ID NO: 880, 881), EP424R (SEQ ID NO: 882, 883), C475L (SEQ ID NO:884, 885), B602L (SEQ ID NO:886, 887), CP530R (SEQ ID NO:888, 889), D339L (SEQ ID NO:890, 891), D117L (SEQ ID NO:863, 865), I243L (SEQID NO: 892, 893), I73R (SEQ ID NO: 894, 895), DP238L (SEQ ID NO: 896, 897), I9R (SEQ ID NO: 898, 899); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 861, 27, 26), NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571); and most preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26).

[0069] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (c), the viral or bacterial vector is selected from the group consisting of: an African swine fever virus vector, an avian pox virus vector, a canine measles virus vector, a herpes virus vector, a varicella virus vector, Lawsonia spp., or Salmonella spp.

[0070] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (c), the viral or bacterial vector contains one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding one, two or more African swine fever virus peptides and / or polypeptides as disclosed and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof.

[0071] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (c), the viral or bacterial vector contains one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, comprising, preferably consisting of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 111), M449R (SEQ ID NO: 112), M450R (SEQ ID NO: 113), M451R (SEQ ID NO: 114), M452R (SEQ ID NO: 115), M453R (SEQ ID NO: 116), M454R (SEQ ID NO: 117), M455R (SEQ ID NO: 118), M456R (SEQ ID NO: 119), M457R (SEQ ID NO: 119), M458R (SEQ ID NO: 119), M459R (SEQ ID NO: 111), M460R (SEQ ID NO: 119), M461R (SEQ ID NO: 119), M462R (SEQ ID NO: 119 NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570), K145R (SEQ ID NO:526, 524, 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO: 468, 466, 465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 489, 487, 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO: 432, 430, 429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ IDNO: 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:733, 732, 731, 730, 729, 728, 727, 726), P1192R (SEQ ID NO:817, 816, 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 692, 691, 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO: 703, 702, 701, 700, 699), EP424R (SEQ ID NO:389, 388, 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO: 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 75, 74, 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 278, 277, 276, 275), D339L (SEQ ID NO: 322, 321, 320), D117L (SEQ ID NO: 862, 864), I243L (SEQ ID NO: 494, 493, 492, 491), I73R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 519, 520), MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 519, 520), MGF505-8R (SEQ ID NO: 519, 520), MGF505-9R (SEQ ID NO: 519, 520), MGF505-10R (SEQ ID NO: 519, 520), MGF505-11 NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ IDNO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25).

[0072] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (c), the viral or bacterial vector contains one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, wherein the one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof contain the following nucleic acid sequence, preferably consist of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO:18, 20, 22, 24, 26, 27, 270, 271, 273, 286, 288, 296, 298, 419, 421, 431, 433, 457, 459, 467 , 469, 480, 482, 488, 490, 517, 519, 525, 527, 567, 569, 571, 573, 718, 720, 723, 725, 773, 775.

[0073] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein according to (c), the viral or bacterial vector contains one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, wherein the one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof contain the following nucleic acid sequence, preferably consist of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to a nucleic acid sequence selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO:857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 859, 298, 296, 288, 286), NO:861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571), K145R (SEQ ID NO:527, 525, 519, 517), B475L (SEQ ID NO:866, 867), H339R (SEQ ID NO: 469, 467, 459, 457), I226R (SEQ ID NO: 490, 488, 482, 480), CP2475L (p37) (SEQ ID NO: 868, 869), CP2475L (p150) (SEQ ID NO: 870, 871), G1211R (SEQ ID NO: 870, 871), NO:433, 431, 421, 419), M1249L (SEQ ID NO:872, 873), MGF505-9R (SEQ ID NO:874, 875), P1192R (SEQ ID NO:876, 877), MGF505-1R (SEQ ID NO:878, 879), MGF505-3R (SEQ ID NO:880, 881), EP424R (SEQ ID NO:882, 883), C475L (SEQ ID NO:884, 885), B602L (SEQ ID NO:886, 887), CP530R (SEQID NO: 888, 889), D339L (SEQ ID NO: 890, 891), D117L (SEQ ID NO: 863, 865), I243L (SEQ ID NO: 892, 893), I73R (SEQ ID NO: 894, 895), DP238L (SEQ ID NO: 896, 897), I9R (SEQ ID NO: 898, 899); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312 ... NO: 861, 273, 271, (MGF505-7R)), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, NO:860, 27, 26).

[0074] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein the African swine fever virus polypeptide, preferably the full-length protein, comprises, preferably consists of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of A238L (SEQ ID NO: 21), A238L (SEQ ID NO: 23), A240L (SEQ ID NO: 853), A240L (SEQ ID NO: 854), B475L (SEQ ID NO: 65), B475L (SEQ ID NO: 66), CP2475 (SEQ ID NO: 256), CP2475 (SEQ ID NO: 257), CP312R (SEQ ID NO: 272), CP312R (SEQ ID NO: 273), NO:274), D1133L (SEQ ID NO:295), D1133L (SEQ ID NO:297), EP402R (SEQ ID NO:378), EP424R (SEQ ID NO:388), EP424R (SEQ ID NO:389), G1211R (SEQ ID NO:430), G1211R (SEQ ID NO:432), H339R (SEQ ID NO:466), H339R (SEQ ID NO:468), I226R (SEQ ID NO:487), I226R (SEQ ID NO:489), K145R (SEQ ID NO:524), K145R (SEQ ID NO:526), ​​M448R (SEQ ID NO:566), M448R (SEQ ID NO:568), M1249L (SEQ ID NO:561), M1249L (SEQ ID NO:562), MGF_100-1L / MGF100-1L (SEQ ID NO:572), MGF505-1R / MGF_505-1R (SEQ ID NO:691), MGF505-1R / MGF_505-1R (SEQ ID NO:692), MGF505-8R / MGF_505-8R (SEQ ID NO:722), MGF505-7R / MGF_505-7R (SEQ ID NO:724), MGF505-8R / MGF_505-8R (SEQ ID NO:772), MGF505-7R / MGF_505-7R (SEQ IDNO: 774), MGF505-9R / MGF_505-9R (SEQ ID NO: 732), MGF505-9R / MGF_505-9R (SEQ ID NO: 733), P1192R (SEQ ID NO: 816), P1192R (SEQ ID NO: 817); and / or wherein the African swine fever virus peptide and / or its immunogenic fragment and / or African swine fever virus polypeptide / full-length protein immunogenic fragment contains the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of A238L (SEQ ID NO: 17), A238L (SEQ ID NO: 19), A240L (SEQ ID NO: 25), B475L (SEQ ID NO: 26), NO:48), B475L (SEQ ID NO:49), B475L (SEQ ID NO:50), B475L (SEQ ID NO:51), B475L (SEQ ID NO:52), B475L (SEQ ID NO:53), B475L (SEQ ID NO:54), B475L (SEQ ID NO:55), B475L (SEQ ID NO:56), B475L (SEQ ID NO:57), B475L (SEQ ID NO:58), B475L (SEQ ID NO:59), B475L (SEQ ID NO:60), B475L (SEQ ID NO:61), B475L (SEQ ID NO:62), B475L (SEQ ID NO:63), B475L (SEQ ID NO:64), CP2475 (SEQ ID NO:235), CP2475 (SEQ ID NO:236), CP2475 (SEQ ID NO:237), CP2475 (SEQ ID NO:238), CP2475 (SEQ ID NO:239), CP2475 (SEQ ID NO:240), CP2475 (SEQ ID NO:241), CP2475 (SEQ ID NO:242), CP2475 (SEQ ID NO:243), CP2475 (SEQ ID NO:244), CP2475 (SEQ ID NO:245), CP2475 (SEQ ID NO:246), CP2475 (SEQ ID NO:247), CP2475 (SEQ ID NO:248), CP2475 (SEQ IDNO:249)、CP2475(SEQ ID NO:250)、CP2475(SEQ ID NO:251)、CP2475(SEQ ID NO:252)、CP2475(SEQID NO:253)、CP2475(SEQ ID NO:254)、CP2475(SEQ ID NO:255)、CP2475L(p37)(SEQ IDNO:261)、CP2475L(p37)(SEQ ID NO:262)、CP2475L(p37)(SEQ ID NO:263)、CP2475L(p37)(SEQ ID NO:264)、CP2475L(p37)(SEQ ID NO:265)、CP2475L(p37)(SEQ ID NO:266)、CP2475L(p150)(SEQ ID NO:258)、CP2475L(p150)(SEQ ID NO:259)、CP2475L(p150)(SEQID NO:260)、CP312R(SEQ ID NO:267)、CP312R(SEQ ID NO:268)、CP312R(SEQ ID NO:269)、D1133L(SEQ ID NO:281)、D1133L(SEQ ID NO:282)、D1133L(SEQ ID NO:283)、D1133L(SEQID NO:284)、D1133L(SEQ ID NO:285)、D1133L(SEQ ID NO:287)、D1133L(SEQ ID NO:289)、D1133L(SEQ ID NO:290)、D1133L(SEQ ID NO:291)、D1133L(SEQ ID NO:292)、D1133L(SEQID NO:293)、D1133L(SEQ ID NO:294)、EP402R(SEQ ID NO:372)、EP402R(SEQ ID NO:373)、EP402R(SEQ ID NO:374)、EP402R(SEQ ID NO:375)、EP402R(SEQ ID NO:376)、EP402R(SEQID NO:377)、EP424R(SEQ ID NO:379)、EP424R(SEQ ID NO:380)、EP424R(SEQ ID NO:381)、EP424R(SEQ ID NO:382)、EP424R(SEQ ID NO:383)、EP424R(SEQ ID NO:384)、EP424R(SEQIDNO:385)、EP424R(SEQ ID NO:386)、EP424R(SEQ ID NO:387)、G1211R(SEQ ID NO:416)、G1211R(SEQ ID NO:417)、G1211R(SEQ ID NO:418)、G1211R(SEQ ID NO:420)、G1211R(SEQID NO:422)、G1211R(SEQ ID NO:423)、G1211R(SEQ ID NO:424)、G1211R(SEQ ID NO:425)、G1211R(SEQ ID NO:426)、G1211R(SEQ ID NO:427)、G1211R(SEQ ID NO:428)、G1211R(SEQID NO:429)、H339R(SEQ ID NO:454)、H339R(SEQ ID NO:455)、H339R(SEQ ID NO:456)、H339R(SEQ ID NO:458)、H339R(SEQ ID NO:460)、H339R(SEQ ID NO:461)、H339R(SEQ IDNO:462)、H339R(SEQ ID NO:463)、H339R(SEQ ID NO:464)、H339R(SEQ ID NO:465)、I226R(SEQ ID NO:478)、I226R(SEQ ID NO:479)、I226R(SEQ ID NO:481)、I226R(SEQ ID NO:483)、I226R(SEQ ID NO:484)、I226R(SEQ ID NO:485)、I226R(SEQ ID NO:486)、K145R(SEQID NO:514)、K145R(SEQ ID NO:515)、K145R(SEQ ID NO:516)、K145R(SEQ ID NO:518)、K145R(SEQ ID NO:520)、K145R(SEQ ID NO:521)、K145R(SEQ ID NO:522)、K145R(SEQ IDNO:523)、M448R(SEQ ID NO:563)、M448R(SEQ ID NO:564)、M448R(SEQ ID NO:565)、M1249L(SEQ ID NO:539)、M1249L(SEQ ID NO:540)、M1249L(SEQ ID NO:541)、M1249L(SEQ ID NO:542)、M1249L(SEQ IDNO:543)、M1249L(SEQ ID NO:544)、M1249L(SEQ ID NO:545)、M1249L(SEQ ID NO:546)、M1249L(SEQ ID NO:547)、M1249L(SEQ ID NO:548)、M1249L(SEQ ID NO:549)、M1249L(SEQ ID NO:550)、M1249L(SEQ ID NO:551)、M1249L(SEQ ID NO:552)、M1249L(SEQ ID NO:553)、M1249L(SEQ ID NO:554)、M1249L(SEQ ID NO:555)、M1249L(SEQ ID NO:556)、M1249L(SEQ ID NO:557)、M1249L(SEQ ID NO:558)、M1249L(SEQ ID NO:559)、M1249L(SEQ ID NO:560)、MGF_100-1L / MGF100-1L(SEQ ID NO:570)、MGF505-1R / MGF_505-1R(SEQID NO:684)、MGF505-1R / MGF_505-1R(SEQ ID NO:685)、MGF505-1R / MGF_505-1R(SEQ IDNO:686)、MGF505-1R / MGF_505-1R(SEQ ID NO:687)、MGF505-1R / MGF_505-1R(SEQ ID NO:688)、MGF505-1R / MGF_505-1R(SEQ ID NO:689)、MGF505-1R / MGF_505-1R(SEQ ID NO:690)、MGF505-8R / MGF_505-8R(SEQ ID NO:717)、MGF505-7R / MGF_505-7R(SEQ ID NO:719)、MGF505-7R / MGF_505-7R(SEQ ID NO:721)、MGF505-9R / MGF_505-9R(SEQ ID NO:726)、MGF505-9R / MGF_505-9R(SEQ ID NO:727)、MGF505-9R / MGF_505-9R(SEQ ID NO:728)、MGF505-9R / MGF_505-9R(SEQ ID NO:729)、MGF505-9R / MGF_505-9R(SEQ ID NO:730)、MGF505-9R / MGF_505-9R(SEQ IDNO:731)、P1192R(SEQ ID NO:801)、P1192R(SEQ ID NO:802)、P1192R(SEQ ID NO:803)、P1192R(SEQ ID NO:804)、P1192R(SEQ ID NO:805)、P1192R(SEQ ID NO:806)、P1192R(SEQ ID NO:807)、P1192R(SEQ ID NO:808)、P1192R(SEQ ID NO:809)、P1192R(SEQ ID NO:810)、P1192R(SEQ ID NO:811)、P1192R(SEQ ID NO:812)、P1192R(SEQ ID NO:813)、P1192R(SEQ ID NO:814)、P1192R(SEQ ID NO:815)。

[0075] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein the African swine fever virus polypeptide, preferably the full-length protein, comprises, preferably consists of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722), M448R (SEQ ID NO: 568, 566), D1133L (SEQ ID NO: 297, 295), CP312R (SEQ ID NO: 274, 272), A240L (SEQ ID NO: 854, 853), A238L (SEQ ID NO: 856), NO: 23, 21), MGF100-1L (SEQ ID NO: 572), K145R (SEQ ID NO: 526, 524), B475L (SEQ ID NO: 66, 65), H339R (SEQ ID NO: 468, 466), I226R (SEQ ID NO: 489, 487), CP2475 (SEQ ID NO: 489, 487), NO:257), CP2475 (SEQ ID NO:256), G1211R (SEQ ID NO:432, 430), M1249L (SEQ ID NO:562, 561), MGF505-9R (SEQ ID NO:733, 732), P1192R (SEQ ID NO:817, 816), MGF505-1R (SEQ ID NO:692, 691), MGF505-3R (SEQ ID NO:703, 702), EP424R (SEQ ID NO:389, 388), C475L (SEQ ID NO:201, 200), B602L (SEQ ID NO:75, 74), CP530R (SEQ ID NO:278, 277), D339L (SEQ ID NO:322, 321), D117L (SEQ ID NO:862, 864), I243L (SEQ ID NO:494, 493), I73R (SEQ ID NO:504, 503), DP238L (SEQ ID NO:327, 326), I9R (SEQ IDNO: 513, 512); and / or wherein the African swine fever virus peptide and / or immunogenic fragment thereof and / or African swine fever virus polypeptide / full-length protein immunogenic fragment comprises the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 721, 719, 717), M448R (SEQ ID NO: 565, 564, 563), D1133L (SEQ ID NO: 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 513, 512); NO:269, 268, 267), A240L (SEQ ID NO:25), A238L (SEQ ID NO:19, 17), MGF100-1L (SEQ ID NO:570), K145R (SEQ ID NO: 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO:465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO:429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ ID NO: 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:731, 730, 729, 728, 727, 726), P1192R (SEQ IDNO: 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO:701, 700, 699), EP424R (SEQ ID NO: 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO:199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 277, 276, 275), D339L (SEQ ID NO: 320), I243L (SEQ ID NO: 492, 491), I73R (SEQ ID NO: 502), DP238L (SEQ ID NO: 325), I9R (SEQ ID NO: 511, 510).

[0076] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein the ASFV polypeptide is a full-length ASFV protein, which is preferably encoded by a polynucleotide sequence comprising, more preferably consisting of: any possible open reading frame (ORF); even more preferably encoded by a polynucleotide sequence comprising, most preferably consisting of: an open reading frame (ORF) with a start codon at the 5' end and a stop codon at the 3' end.

[0077] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided, wherein the immunogenicity (and / or immune response) of the immunogenic composition or vaccine or pharmaceutical composition or any immunogenic fragment contained therein is indicated / characterized by an induced IFN-γ response, preferably in a porcine IFN-γ ELISpot assay, more preferably in a porcine IFN-γ ELISpot assay as described in Example 1.

[0078] In yet another specific aspect, an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein is provided for use in a method for reducing or preventing clinical symptoms or diseases caused by infection with at least one African swine fever virus, preferably a pathogenic African swine fever virus, in porcine animals, preferably pigs, or for use in a method for treating and / or preventing infection with at least one African swine fever virus, preferably a pathogenic African swine fever virus, wherein the clinical symptoms or diseases caused by infection with at least one African swine fever virus, preferably a pathogenic African swine fever virus, or the at least one African swine fever virus, preferably a pathogenic African swine fever virus infection is preferably selected from the group consisting of African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin mottling, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia. The present invention is also intended to include corresponding methods for reducing or preventing clinical symptoms or diseases caused by infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, or corresponding methods for treating and / or preventing infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus (which comprises administering to the porcine animals, preferably pigs, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein), as well as corresponding uses of the immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for reducing or preventing clinical symptoms or diseases caused by infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus, or for treating and / or preventing infection of porcine animals, preferably pigs, by at least one African swine fever virus, preferably a pathogenic African swine fever virus.

[0079] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided for use in a method for immunizing a porcine animal, preferably a pig, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a porcine animal, preferably a pig, the method comprising the steps of administering an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein to a porcine animal, preferably a pig, wherein the immunogenic composition or vaccine or pharmaceutical composition does not cause clinical symptoms of infection but is capable of inducing the porcine animal, Preferably, it is an immune response of pigs to the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus, wherein the clinical disease or the clinical infection symptoms are preferably selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, thrombocytopenia. The present invention is also intended to include a corresponding method for immunizing a porcine animal, preferably a pig, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a porcine animal, preferably a pig, comprising the steps of administering to a porcine animal, preferably a pig, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein, wherein the immunogenic composition or vaccine or pharmaceutical composition does not cause clinical symptoms of infection but is capable of inducing immunity of the porcine animal, preferably a pig, to the at least one African swine fever virus, preferably the pathogenicity of the at least one African swine fever virus. and the corresponding use of an immunogenic composition, vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for immunizing porcine animals, preferably pigs, against clinical disease caused by at least one African swine fever virus, preferably pathogenic African swine fever virus, in porcine animals, preferably pigs, wherein the immunogenic composition, vaccine or pharmaceutical composition does not cause clinical symptoms of infection but can induce an immune response that immunizes porcine animals, preferably pigs, against the at least one African swine fever virus, preferably the at least one pathogenic form of African swine fever virus.

[0080] In yet another specific aspect, an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein is provided for use in a method for prime-boost immunization of a porcine animal, preferably a pig, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in a porcine animal, preferably a pig, the method comprising the steps of administering to the porcine animal, preferably a pig, once or twice an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein containing: (i) as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof; or (ii) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, which contains as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof (prime boost) step); and subsequently - after one or two primings - administering a live attenuated African swine fever virus, preferably BA71ΔCD2, to a porcine animal, preferably a pig (boosting step); wherein the immunogenic composition or vaccine or pharmaceutical composition and the live attenuated African swine fever virus independently of each other do not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal, preferably the pig, against the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus, wherein the clinical disease or the clinical infection symptoms are preferably selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin mottling, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleeds, abortion, leukopenia, thrombocytopenia.The present invention is also intended to include a method for prime-boost immunization of porcine animals, preferably pigs, against clinical disease caused by at least one African swine fever virus, preferably a pathogenic African swine fever virus, in porcine animals, preferably pigs, the method comprising the following steps: administering to porcine animals, preferably pigs, once or twice an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein containing: (i) as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof; or (ii) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, which contains as the sole immunogenic component one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof and subsequently - after one or two primings - administering a live attenuated African swine fever virus, preferably BA71ΔCD2, to a porcine animal, preferably a pig (boosting step); wherein the immunogenic composition or vaccine or pharmaceutical composition and the live attenuated African swine fever virus independently of each other do not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal, preferably the pig, against the at least one African swine fever virus, preferably the pathogenic form of the at least one African swine fever virus; and the corresponding use of the immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein for the preparation of a medicament for prime-boost immunization of porcine animals, preferably pigs, against clinical diseases caused by at least one African swine fever virus, preferably pathogenic African swine fever virus in porcine animals, preferably pigs.

[0081] In yet another specific aspect, a kit is provided for vaccinating porcine animals, preferably pigs, against diseases associated with at least one African swine fever virus (preferably a pathogenic African swine fever virus) and / or reducing the incidence or severity of one or more clinical symptoms associated with at least one African swine fever virus (preferably a pathogenic African swine fever virus) or caused by at least one African swine fever virus (preferably a pathogenic African swine fever virus) in porcine animals, preferably pigs, the kit comprising:

[0082] (a) a dispenser capable of administering the vaccine to the porcine animal; and

[0083] (b) an immunogenic composition or vaccine or pharmaceutical composition as described and / or claimed herein; and

[0084] (c) optional instruction booklet;

[0085] The disease or clinical symptoms are preferably selected from the group consisting of African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival secretions, bleeding, nosebleed, abortion, leukopenia, and thrombocytopenia.

[0086] In yet another specific aspect, African swine fever virus peptides and / or polypeptides are provided, preferably full-length proteins and / or immunogenic fragments thereof, comprising, preferably consisting of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,19,21,23,25,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,4 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、272、274、275、276、277、278、279、280、281、282、283、284、285、287、289、290、291、292、293、294、295、297、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、420、422、423、424、425、426、427、428、429、430、432、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、458、460、461、462、463、464、465、466、468、470、471、472、473、474、475、476、477、478、479、481、483、484、485、486、487、489、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、518、520、521、522、523、524、526、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、568、570、572、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、719、721、722、724、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、774、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854。、

[0087] In yet another specific aspect, African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, are provided, which contain, preferably consist of, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 569, 571, 572, 573, 574, 575), NO:297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570), K145R (SEQ ID NO:526, 524, 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO: 468, 466, 465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 489, 487, 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO: 432, 430, 429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ ID NO: 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ IDNO:733, 732, 731, 730, 729, 728, 727, 726), P1192R (SEQ ID NO:817, 816, 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 692, 691, 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO: 703, 702, 701, 700, 699), EP424R (SEQ ID NO:389, 388, 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO: 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 75, 74, 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 278, 277, 276, 275), D339L (SEQ ID NO: 322, 321, 320), D117L (SEQ ID NO: 862, 864), I243L (SEQ ID NO: 494, 493, 492, 491), I73R (SEQ ID NO: 504, 503, 502), DP238L (SEQ ID NO: 327, 326, 325), I9R (SEQ ID NO: 513, 512, 511, 510); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 568, 566, 565, 564, 563), D1133L (SEQ ID NO: 513), MGF505-7R / MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), MGF505-8R (SEQ ID NO: 513), NO: 297, 295, 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 274, 272, 269, 268, 267), A240L (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO: 854, 853, 25), A238L (SEQ ID NO: 23, 21, 19, 17), MGF100-1L (SEQ ID NO: 572, 570); and most preferably is selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722, 721, 719, 717), M448R (SEQ ID NO:ID NO:568、566、565、564、563)、D1133L(SEQ ID NO:297、295、294、293、292、291、290、289、287、285、284、283、282、281)、CP312R(SEQ ID NO:274、272、269、268、267)、A240L(SEQID NO:854、853、25)。

[0088] In yet another specific aspect, African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, are provided, wherein the African swine fever virus polypeptide, preferably the full-length protein, contains the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of: A238L (SEQ ID NO: 21), A238L (SEQ ID NO: 23), A240L (SEQ ID NO: 853), A240L (SEQ ID NO: 854), B475L (SEQ ID NO: 65), B475L (SEQ ID NO: 66), CP2475 (SEQ ID NO: 256), CP2475 (SEQ ID NO: 257), CP312R (SEQ ID NO: 111), CP312R (SEQ ID NO: 112), CP312R (SEQ ID NO: 113), CP312R (SEQ ID NO: 114), CP312R (SEQ ID NO: 115), CP312R (SEQ ID NO: 116), CP312R (SEQ ID NO: 117), CP312R (SEQ ID NO: 118), CP312R (SEQ ID NO: 119), CP312R (SEQ ID NO: 120), CP312R (SEQ ID NO: 121), CP312R (SEQ ID NO: 122), CP312R (SEQ ID NO: 123), NO:272), CP312R (SEQ ID NO:274), D1133L (SEQ ID NO:295), D1133L (SEQ ID NO:297), EP402R (SEQ ID NO:378), EP424R (SEQ ID NO:388), EP424R (SEQ ID NO:389), G1211R (SEQ ID NO:430), G1211R (SEQ ID NO:432), H339R (SEQ ID NO:466), H339R (SEQ ID NO:468), I226R (SEQ ID NO:487), I226R (SEQ ID NO:489), K145R (SEQ ID NO:524), K145R (SEQ ID NO:526), ​​M448R (SEQ ID NO:566), M448R (SEQ ID NO:568), M1249L (SEQ ID NO:561), M1249L (SEQ ID NO:562), MGF_100-1L / MGF100-1L (SEQ ID NO:572), MGF505-1R / MGF_505-1R (SEQ ID NO:691), MGF505-1R / MGF_505-1R (SEQ ID NO:692), MGF505-8R / MGF_505-8R (SEQ ID NO:722), MGF505-7R / MGF_505-7R (SEQ ID NO:724), MGF505-8R / MGF_505-8R (SEQ ID NO:772), MGF505-7R / MGF_505-7R (SEQ IDNO: 774), MGF505-9R / MGF_505-9R (SEQ ID NO: 732), MGF505-9R / MGF_505-9R (SEQ ID NO: 733), P1192R (SEQ ID NO: 816), P1192R (SEQ ID NO: 817); and / or wherein the African swine fever virus peptide and / or its immunogenic fragment and / or African swine fever virus polypeptide / full-length protein immunogenic fragment contains the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of A238L (SEQ ID NO: 17), A238L (SEQ ID NO: 19), A240L (SEQ ID NO: 25), B475L (SEQ ID NO: 26), NO:48), B475L (SEQ ID NO:49), B475L (SEQ ID NO:50), B475L (SEQ ID NO:51), B475L (SEQ ID NO:52), B475L (SEQ ID NO:53), B475L (SEQ ID NO:54), B475L (SEQ ID NO:55), B475L (SEQ ID NO:56), B475L (SEQ ID NO:57), B475L (SEQ ID NO:58), B475L (SEQ ID NO:59), B475L (SEQ ID NO:60), B475L (SEQ ID NO:61), B475L (SEQ ID NO:62), B475L (SEQ ID NO:63), B475L (SEQ ID NO:64), CP2475 (SEQ ID NO:235), CP2475 (SEQ ID NO:236), CP2475 (SEQ ID NO:237), CP2475 (SEQ ID NO:238), CP2475 (SEQ ID NO:239), CP2475 (SEQ ID NO:240), CP2475 (SEQ ID NO:241), CP2475 (SEQ ID NO:242), CP2475 (SEQ ID NO:243), CP2475 (SEQ ID NO:244), CP2475 (SEQ ID NO:245), CP2475 (SEQ ID NO:246), CP2475 (SEQ ID NO:247), CP2475 (SEQ ID NO:248), CP2475 (SEQ IDNO:249)、CP2475(SEQ ID NO:250)、CP2475(SEQ ID NO:251)、CP2475(SEQ ID NO:252)、CP2475(SEQID NO:253)、CP2475(SEQ ID NO:254)、CP2475(SEQ ID NO:255)、CP2475L(p37)(SEQ IDNO:261)、CP2475L(p37)(SEQ ID NO:262)、CP2475L(p37)(SEQ ID NO:263)、CP2475L(p37)(SEQ ID NO:264)、CP2475L(p37)(SEQ ID NO:265)、CP2475L(p37)(SEQ ID NO:266)、CP2475L(p150)(SEQ ID NO:258)、CP2475L(p150)(SEQ ID NO:259)、CP2475L(p150)(SEQID NO:260)、CP312R(SEQ ID NO:267)、CP312R(SEQ ID NO:268)、CP312R(SEQ ID NO:269)、D1133L(SEQ ID NO:281)、D1133L(SEQ ID NO:282)、D1133L(SEQ ID NO:283)、D1133L(SEQID NO:284)、D1133L(SEQ ID NO:285)、D1133L(SEQ ID NO:287)、D1133L(SEQ ID NO:289)、D1133L(SEQ ID NO:290)、D1133L(SEQ ID NO:291)、D1133L(SEQ ID NO:292)、D1133L(SEQID NO:293)、D1133L(SEQ ID NO:294)、EP402R(SEQ ID NO:372)、EP402R(SEQ ID NO:373)、EP402R(SEQ ID NO:374)、EP402R(SEQ ID NO:375)、EP402R(SEQ ID NO:376)、EP402R(SEQID NO:377)、EP424R(SEQ ID NO:379)、EP424R(SEQ ID NO:380)、EP424R(SEQ ID NO:381)、EP424R(SEQ ID NO:382)、EP424R(SEQ ID NO:383)、EP424R(SEQ ID NO:384)、EP424R(SEQIDNO:385)、EP424R(SEQ ID NO:386)、EP424R(SEQ ID NO:387)、G1211R(SEQ ID NO:416)、G1211R(SEQ ID NO:417)、G1211R(SEQ ID NO:418)、G1211R(SEQ ID NO:420)、G1211R(SEQID NO:422)、G1211R(SEQ ID NO:423)、G1211R(SEQ ID NO:424)、G1211R(SEQ ID NO:425)、G1211R(SEQ ID NO:426)、G1211R(SEQ ID NO:427)、G1211R(SEQ ID NO:428)、G1211R(SEQID NO:429)、H339R(SEQ ID NO:454)、H339R(SEQ ID NO:455)、H339R(SEQ ID NO:456)、H339R(SEQ ID NO:458)、H339R(SEQ ID NO:460)、H339R(SEQ ID NO:461)、H339R(SEQ IDNO:462)、H339R(SEQ ID NO:463)、H339R(SEQ ID NO:464)、H339R(SEQ ID NO:465)、I226R(SEQ ID NO:478)、I226R(SEQ ID NO:479)、I226R(SEQ ID NO:481)、I226R(SEQ ID NO:483)、I226R(SEQ ID NO:484)、I226R(SEQ ID NO:485)、I226R(SEQ ID NO:486)、K145R(SEQID NO:514)、K145R(SEQ ID NO:515)、K145R(SEQ ID NO:516)、K145R(SEQ ID NO:518)、K145R(SEQ ID NO:520)、K145R(SEQ ID NO:521)、K145R(SEQ ID NO:522)、K145R(SEQ IDNO:523)、M448R(SEQ ID NO:563)、M448R(SEQ ID NO:564)、M448R(SEQ ID NO:565)、M1249L(SEQ ID NO:539)、M1249L(SEQ ID NO:540)、M1249L(SEQ ID NO:541)、M1249L(SEQ ID NO:542)、M1249L(SEQ IDNO:543)、M1249L(SEQ ID NO:544)、M1249L(SEQ ID NO:545)、M1249L(SEQ ID NO:546)、M1249L(SEQ ID NO:547)、M1249L(SEQ ID NO:548)、M1249L(SEQ ID NO:549)、M1249L(SEQ ID NO:550)、M1249L(SEQ ID NO:551)、M1249L(SEQ ID NO:552)、M1249L(SEQ ID NO:553)、M1249L(SEQ ID NO:554)、M1249L(SEQ ID NO:555)、M1249L(SEQ ID NO:556)、M1249L(SEQ ID NO:557)、M1249L(SEQ ID NO:558)、M1249L(SEQ ID NO:559)、M1249L(SEQ ID NO:560)、MGF_100-1L / MGF100-1L(SEQ ID NO:570)、MGF505-1R / MGF_505-1R(SEQID NO:684)、MGF505-1R / MGF_505-1R(SEQ ID NO:685)、MGF505-1R / MGF_505-1R(SEQ IDNO:686)、MGF505-1R / MGF_505-1R(SEQ ID NO:687)、MGF505-1R / MGF_505-1R(SEQ ID NO:688)、MGF505-1R / MGF_505-1R(SEQ ID NO:689)、MGF505-1R / MGF_505-1R(SEQ ID NO:690)、MGF505-8R / MGF_505-8R(SEQ ID NO:717)、MGF505-7R / MGF_505-7R(SEQ ID NO:719)、MGF505-7R / MGF_505-7R(SEQ ID NO:721)、MGF505-9R / MGF_505-9R(SEQ ID NO:726)、MGF505-9R / MGF_505-9R(SEQ ID NO:727)、MGF505-9R / MGF_505-9R(SEQ ID NO:728)、MGF505-9R / MGF_505-9R(SEQ ID NO:729)、MGF505-9R / MGF_505-9R(SEQ ID NO:730)、MGF505-9R / MGF_505-9R(SEQ IDNO:731)、P1192R(SEQ ID NO:801)、P1192R(SEQ ID NO:802)、P1192R(SEQ ID NO:803)、P1192R(SEQ ID NO:804)、P1192R(SEQ ID NO:805)、P1192R(SEQ ID NO:806)、P1192R(SEQ ID NO:807)、P1192R(SEQ ID NO:808)、P1192R(SEQ ID NO:809)、P1192R(SEQ ID NO:810)、P1192R(SEQ ID NO:811)、P1192R(SEQ ID NO:812)、P1192R(SEQ ID NO:813)、P1192R(SEQ ID NO:814)、P1192R(SEQ ID NO:815)。

[0089] In yet another specific aspect, African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, are provided, wherein the African swine fever virus polypeptide, preferably the full-length protein, contains the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 774, 772, 724, 722), M448R (SEQ ID NO: 568, 566), D1133L (SEQ ID NO: 297, 295), CP312R (SEQ ID NO: 274, 272), A240L (SEQ ID NO: 854, 853), A238L (SEQ ID NO: 856), NO: 23, 21), MGF100-1L (SEQ ID NO: 572), K145R (SEQ ID NO: 526, 524), B475L (SEQ ID NO: 66, 65), H339R (SEQ ID NO: 468, 466), I226R (SEQ ID NO: 489, 487), CP2475 (SEQ ID NO: 489, 487), NO:257), CP2475 (SEQ ID NO:256), G1211R (SEQ ID NO:432, 430), M1249L (SEQ ID NO:562, 561), MGF505-9R (SEQ ID NO:733, 732), P1192R (SEQ ID NO:817, 816), MGF505-1R (SEQ ID NO:692, 691), MGF505-3R (SEQ ID NO:703, 702), EP424R (SEQ ID NO:389, 388), C475L (SEQ ID NO:201, 200), B602L (SEQ ID NO:75, 74), CP530R (SEQ ID NO:278, 277), D339L (SEQ ID NO:322, 321), D117L (SEQ ID NO:862, 864), I243L (SEQ ID NO:494, 493), I73R (SEQ ID NO:504, 503), DP238L (SEQ ID NO:327, 326), I9R (SEQ IDNO: 513, 512); and / or wherein the African swine fever virus peptide and / or immunogenic fragment thereof and / or African swine fever virus polypeptide / full-length protein immunogenic fragment comprises the following amino acid sequence, preferably consists of the following amino acid sequence: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 721, 719, 717), M448R (SEQ ID NO: 565, 564, 563), D1133L (SEQ ID NO: 294, 293, 292, 291, 290, 289, 287, 285, 284, 283, 282, 281), CP312R (SEQ ID NO: 513, 512); NO:269, 268, 267), A240L (SEQ ID NO:25), A238L (SEQ ID NO:19, 17), MGF100-1L (SEQ ID NO:570), K145R (SEQ ID NO: 523, 522, 521, 520, 518, 516, 515, 514), B475L (SEQ ID NO: 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48), H339R (SEQ ID NO:465, 464, 463, 462, 461, 460, 458, 456, 455, 454), I226R (SEQ ID NO: 486, 485, 484, 483, 481, 479, 478), CP2475L (p37) (SEQ ID NO: 266, 265, 264, 263, 262, 261), CP2475L (p150) (SEQ ID NO: 260, 259, 258), G1211R (SEQ ID NO:429, 428, 427, 426, 425, 424, 423, 422, 420, 418, 417, 416), M1249L (SEQ ID NO: 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539), MGF505-9R (SEQ ID NO:731, 730, 729, 728, 727, 726), P1192R (SEQ IDNO: 815, 814, 813, 812, 811, 810, 809, 808, 807, 806, 805, 804, 803, 802, 801), MGF505-1R (SEQ ID NO: 690, 689, 688, 687, 686, 685, 684), MGF505-3R (SEQ ID NO:701, 700, 699), EP424R (SEQ ID NO: 387, 386, 385, 384, 383, 382, ​​381, 380, 379), C475L (SEQ ID NO:199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188), B602L (SEQ ID NO: 73, 72, 71, 70, 69, 68, 67), CP530R (SEQ ID NO: 277, 276, 275), D339L (SEQ ID NO: 320), I243L (SEQ ID NO: 492, 491), I73R (SEQ ID NO: 502), DP238L (SEQ ID NO: 325), I9R (SEQ ID NO: 511, 510).

[0090] In yet another specific aspect, African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof are provided that encode African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof.

[0091] In yet another specific aspect, an African swine fever virus oligonucleotide and / or polynucleotide and / or immunogenic fragment thereof encoding an African swine fever virus peptide and / or polypeptide, preferably a full-length protein and / or an immunogenic fragment thereof is provided, which contains the following nucleic acid sequence, preferably consists of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with a nucleic acid sequence selected from the group consisting of: SEQ ID NO:18, 20, 22, 24, 26, 27, 270, 271, 273, 286, 288, 296, 298, 419, 421, 431, 433, 457, 459, 467 , 469, 480, 482, 488, 490, 517, 519, 525, 527, 567, 569, 571, 573, 718, 720, 723, 725, 773, 775.

[0092] In yet another specific aspect, an African swine fever virus oligonucleotide and / or polynucleotide and / or immunogenic fragment thereof encoding an African swine fever virus peptide and / or polypeptide, preferably a full-length protein and / or an immunogenic fragment thereof is provided, which contains the following nucleic acid sequence, preferably consists of the following nucleic acid sequence: a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with a nucleic acid sequence selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: ... ID NO:861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 573, 571), K145R (SEQ ID NO: 527, 525, 519, 517), B475L (SEQ ID NO: 866, 867), H339R (SEQ ID NO: 469, 467, 459, 457), I226R (SEQ ID NO: 490, 488, 482, 480), CP2475L (p37) (SEQ ID NO:868, 869), CP2475L (p150) (SEQ ID NO:870, 871), G1211R (SEQ ID NO:433, 431, 421, 419), M1249L (SEQ ID NO:872, 873), MGF505-9R (SEQ ID NO:874, 875), P1192R (SEQ ID NO:876, 877), MGF505-1R (SEQ ID NO:874, 875) NO:878, 879), MGF505-3R (SEQ ID NO:880, 881), EP424R (SEQ ID NO:882, 883), C475L (SEQ ID NO:884, 885), B602L (SEQ ID NO:886, 887), CP530R (SEQ ID NO:886, 887), NO:888, 889), D339L (SEQ ID NO:890, 891), D117L (SEQ ID NO:863, 865), I243L (SEQ ID NO:892, 893), I73R (SEQ IDNO: 894, 895), DP238L (SEQ ID NO: 896, 897), I9R (SEQ ID NO: 898, 899); preferably selected from the group consisting of MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26), A238L (SEQ ID NO: 24, 22, 20, 18), MGF100-1L (SEQ ID NO: 861, 273), NO: 573, 571); and most preferably is selected from the group consisting of: MGF505-7R / MGF505-8R (SEQ ID NO: 857, 775, 773, 725, 723, 720, 718), M448R (SEQ ID NO: 858, 569, 567), D1133L (SEQ ID NO: 859, 298, 296, 288, 286), CP312R (SEQ ID NO: 861, 273, 901, 900, 271, 270), A240L (SEQ ID NO: 860, 27, 26).

[0093] In yet another specific aspect, provided are African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof, or African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof, wherein the ASFV polypeptide is an ASFV full-length protein, and the ASFV full-length protein is preferably encoded by a polynucleotide sequence comprising, more preferably consisting of: any possible open reading frame (ORF); even more preferably, encoded by a polynucleotide sequence comprising, most preferably consisting of: an open reading frame (ORF) with a start codon at the 5' end and a stop codon at the 3' end.

[0094] In yet another specific aspect, African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof, or African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides as described and / or claimed herein, preferably full-length proteins and / or immunogenic fragments thereof, are provided, wherein the immunogenicity (and / or immune response) of the immunogenic composition or vaccine or pharmaceutical composition or any immunogenic fragment contained therein is indicated / characterized by an induced IFN-γ response, preferably by an induced IFN-γ ELISpot assay in porcine animals, more preferably in a porcine IFN-γ ELISpot assay as described in Example 1.

[0095] In yet another specific aspect, a vector is provided containing one, two, three or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof as described and / or claimed herein. The vector preferably contains three African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof, and the three African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof are preferably selected from EP402R, CP312R and A240L (multiple epitopes-1, ME-1), more preferably contains the following, most preferably consists of the following: NO:855 consisting of a nucleic acid sequence; or containing thirteen African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof, the thirteen African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof are preferably selected from D1133L, G1211R, M1249L, MGF505-9R, P1192R, CP2475L (p150), B475L, EP424R, H339R, I226R, K145R, MGF505-1R and CP2475L (p37) (multiple epitopes-II, ME-II), more preferably containing the following, most preferably consisting of the following: a nucleic acid sequence selected from the group consisting of SEQ ID NO:856.

[0096] In yet another specific aspect, there is provided a host cell, preferably a mammalian host cell, containing a vector as described and / or claimed herein.

[0097] definition

[0098] Unless otherwise defined, all technical and scientific terms used herein at the time of application have the same meaning as commonly understood by those skilled in the art to which the invention pertains. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Unless otherwise stated, the use of "or" herein means "and / or". In addition, the use of the term "including" and other forms such as "includes / included" is not restrictive. All patents and publications mentioned herein are incorporated herein by reference.

[0099] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of virology, molecular biology, microbiology, recombinant DNA technology, protein chemistry, and immunology, which are within the skill of the art and are fully explained in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, 2nd ed. (1989); DNA Cloning, Vols. I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, ed., 1984); Animal Cell Culture (R. K. Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods in Enzymology series (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Protein purification methods - a practical approach (E. L. V. Harris and S. Angal, eds., IRL Press of Oxford University Press); and Handbook of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications).

[0100] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific DNA, so the polypeptide sequence or program parameters can certainly vary. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments of the present invention, and are not intended to be restrictive. It must be noted that, unless the context clearly indicates otherwise, the singular forms "a / an" and "the / such" used in this specification and the appended claims include plural references. Therefore, for example, reference to "antigen" includes a mixture of two or more antigens, and reference to "excipient" includes a mixture of two or more excipients, and the like.

[0101] The term "viral or bacterial vector" describes a genetically modified virus or bacterium that has been manipulated using recombinant DNA technology in such a way that its entry into a host cell causes the vector to carry out a specific biological activity, for example, expression of a transgene such as an ASFV gene. In one specific aspect, the transgene is an ASFV antigen. Viral or bacterial vectors may be replicable or non-replicable in the target cell, tissue, or organism. In this context, the terms "viral vector" and "virus" are used interchangeably, as are the terms "bacterial vector" and "bacteria."

[0102] Production of viral or bacterial vectors can be achieved using any suitable genetic engineering technique well known in the art, including, but not limited to, standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, DNA sequencing, and transfection in cell culture, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)) or K. Maramorosch and H. Koprowski (Methods in Virology Volume VIII, Academic Press Inc. London, UK (2014)).

[0103] A viral or bacterial vector may include coding regions for two or more proteins of interest. For example, a viral or bacterial vector may include a coding region for a first protein of interest and a coding region for a second protein of interest. The first protein of interest and the second protein of interest may be the same or different. In some embodiments, a viral or bacterial vector may include coding regions for a third or fourth protein of interest. The third and fourth proteins of interest may be the same or different. The total length of the two or more proteins of interest encoded by a viral or bacterial vector may vary. For example, the total length of the two or more proteins of interest may be at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 550 amino acids, at least about 600 amino acids, at least about 650 amino acids, at least about 700 amino acids, at least about 750 amino acids, at least about 800 amino acids, or longer.

[0104] According to a specific aspect of the invention, the term "viral or bacterial vector" or alternatively "viral or bacterial construct" refers to a recombinant virus or bacterial construct derived from a virus or bacterium, which is selected from the group consisting of: African swine fever virus vector, avian pox virus vector, canine measles virus vector, herpes virus vector, varicella virus vector, Lawsonia spp., Salmonella spp.

[0105] The terms "viral or bacterial vector" and "viral or bacterial construct" are used interchangeably.

[0106] As used herein, the term "construct" refers to a recombinant nucleic acid such as a plasmid, a BAC, or an artificially generated recombinant virus or bacteria.

[0107] The term "plasmid" refers to cytoplasmic DNA that replicates independently of the bacterial chromosome within a bacterial host cell. In one specific aspect of the present invention, the terms "plasmid" and / or "transfer plasmid" refer to components of recombinant DNA technology that can be used, for example, to construct expression cassettes for insertion into viral vectors. In another specific aspect, the term "plasmid" can be used to designate a plasmid that can be used for DNA vaccination purposes.

[0108] As used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to any nucleic acid.

[0109] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "RNA sequence," "cDNA sequence," or "DNA sequence" refer to oligonucleotides, nucleotides, or polynucleotides, as well as fragments and portions thereof, and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent either the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of the two. The nucleic acid sequences of the present invention may be prepared using standard techniques well known to those skilled in the art.

[0110] The terms "nucleic acid," "nucleic acid sequence," and "nucleotide sequence" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0111] The term "complementary nucleotide sequence" describes one of two paired strands of a polynucleotide such as DNA or RNA. The nucleotide sequence of the complementary strand mirrors the nucleotide sequence of its paired strand such that for each adenylate, it contains a thymine (or for RNA, it contains a uracil), for each guanine, it contains a cytosine, and vice versa. For example, the complementary nucleotide sequence of 5'-GCATAC-3' is 3'-CGTATG-5', or for RNA, the complementary nucleotide sequence is 3'-CGUAUG-5'.

[0112] As used herein, the term "expression" refers to the transcription and / or translation of a nucleic acid sequence in a host cell. According to a particular aspect of the present invention, the term "expression" refers to the transcription and / or translation of a heterologous and / or exogenous nucleic acid sequence in a host cell. The expression level of the desired product in the host cell can be determined based on the amount of the corresponding RNA or mRNA present in the cell or the amount of the desired polypeptide encoded by the selected sequence. For example, the mRNA transcribed from the selected sequence can be quantitatively analyzed by northern blot hybridization, ribonuclease RNA protection, in situ hybridization with cellular RNA, or by RTqPCR (reverse transcription followed by quantitative PCR). The protein expressed from the selected sequence can be quantitatively analyzed by various methods, such as by ELISA, by immunoblotting, by radioimmunoassay, by immunoprecipitation, by protein bioactivity analysis, or by protein immunostaining followed by FACS analysis.

[0113] The term "viral load" is well known to those skilled in the art. In this article, the term viral load can be used interchangeably with the term "viral titer". Viral load or viral titer is a measure of the severity of an active viral infection and can be determined by methods known to those skilled in the art. Determination can be based on viral protein detection and further detection such as by antibody binding to viral proteins, or alternatively, by viral nucleic acid detection using amplification methods such as RT-PCR. Monitoring virion-associated viral RNA in plasma by nucleic acid amplification methods is a parameter widely used to assess the status and development of retroviral diseases and for assessing the effectiveness of preventive and therapeutic interventions. For example, viral load or viral titer can be calculated by estimating the amount of live virus in the body fluid involved, such as the number of RNA copies per milliliter of plasma. Preferably, the term "viral load" or "viral titer" is a measure of infectious units per volume of viral preparation. Viral titer is the endpoint of a biological procedure and is defined as the dilution when a specific ratio of tests performed in parallel show an effect. Specifically, the tissue culture infectious dose fifty per milliliter (TCID50 / ml) gives the dilution of a viral preparation at which 50% of multiple cell cultures inoculated in parallel are infected, the parallel inoculations being performed at that dilution.

[0114] By definition, when each nucleotide sequence or each gene inserted into a host cell and thereby encoding a separate protein or RNA comes from a different (viral or bacterial) species, it is called "exogenous", "exogenous sequence", "exogenous gene", "exogenous coding sequence", "exogenous antigen encoding sequence" relative to the host cell.

[0115] By definition, each nucleotide sequence or each gene that is inserted into a host cell and thereby encodes a respective protein or RNA is referred to as "heterologous," "heterologous sequence," "heterologous gene," "heterologous coding sequence," "transgene," or "heterologous protein" with respect to the host cell. This applies even if the sequence or gene to be introduced is identical to an endogenous sequence or endogenous gene of the host cell. For example, a specific promoter sequence introduced into a viral or bacterial vector at a different site or in a modified form compared to the wild-type virus or bacterium is by definition a heterologous sequence. As used herein, with respect to a sequence or gene of interest, such as an antigen, the term "heterologous" means that the sequence or gene of interest, in particular the antigen, is expressed outside of its native subspecies.

[0116] The term "non-naturally occurring" means any sequence of interest, such as an antigen, or gene that is not naturally occurring in this context (such as a hybridizing sequence), or a sequence of interest, such as an antigen, or gene that is from a different species, or a sequence of interest, such as an antigen, or gene that is not a product of nature due to artificial mutation, insertion, deletion, or the like.

[0117] Throughout the present specification, the term "recombinant" is used interchangeably with the terms "non-naturally occurring," "heterologous," and "exogenous." Thus, a "recombinant" protein is a protein expressed from a heterologous or exogenous nucleotide sequence. The term recombinant, as used with respect to a virus or bacterium, means a virus or bacterium produced by artificial manipulation of the viral or bacterial genome. A virus or bacterium containing a heterologous or exogenous sequence, such as an exogenous antigen encoding sequence, is a recombinant virus or bacterium. The term recombinant virus or bacterium and the term non-naturally occurring virus or bacterium are used interchangeably.

[0118] Thus, the term "heterologous vector" means a vector containing a heterologous or exogenous nucleotide sequence. The term "recombinant vector" means a vector containing a heterologous or recombinant nucleotide sequence.

[0119] As used herein, "sequence homology" refers to a method for determining the relatedness of two sequences. To determine sequence homology, two or more sequences are most preferably aligned and, if necessary, gaps are introduced. However, when determining sequence homology, in contrast to "sequence identity", conservative amino acid substitutions are considered matches. In other words, to obtain a polypeptide or polynucleotide with 95% sequence homology to a reference sequence, 85%, preferably 90%, 91%, 92%, 93%, 94%, even more preferably 95%, 96%, 97%, 98%, 99%, 99.9% of the amino acid residues or nucleotides in the reference sequence must match or contain conservative substitutions of another amino acid or nucleotide, or a certain number of amino acids or nucleotides of up to 15%, preferably up to 10%, 9%, 8%, 7%, 6%, even more preferably up to 5%, 4%, 3%, 2%, 1%, 0.1% of the total amino acid residues or nucleotides in the reference sequence (excluding conservative substitutions) may be inserted into the reference sequence. Preferably, the homologous sequence contains a stretch of at least 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[0120] As known in the art, "sequence identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., a reference sequence and a given sequence to be compared to the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between the sequence strings. Following this alignment, sequence identity is determined on a position-by-position basis, e.g., if the nucleotides or amino acid residues are identical at a particular position, the sequences are "identical" at that position. The total number of such positional identities is then divided by the total number of nucleotides or residues in the reference sequence to yield the percent sequence identity. Sequence identity can be readily calculated by known methods, including, but not limited to, Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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 for determining sequence identity are designed to give the maximum match between the sequences tested. Methods for determining sequence identity are encoded in publicly available computer programs that 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, SF et al., J. Molec. Biol., 215: 403-410 (1990)).The BLASTX program is publicly available from NCBI and other sources (BLASTX Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs use default gap weights to optimally align sequences to produce the highest level of sequence identity between a given sequence and a reference sequence. By way of illustration, by a polynucleotide having a nucleotide sequence that has at least, for example, 85%, preferably 90%, 91%, 92%, 93%, 94%, even more preferably 95%, 96%, 97%, 98%, 99%, 99.9% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of a 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 for every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that is at least 85%, preferably 90%, 91%, 92%, 93%, 94%, even more preferably 95%, 96%, 97%, 98%, 99%, 99.9% identical to a reference nucleotide sequence, up to 15%, preferably 10%, 9%, 8%, 7%, 6%, even more preferably 5%, 4%, 3%, 2%, 1%, 0.1% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a certain number of nucleotides up to 15%, preferably 10%, 9%, 8%, 7%, 6%, even more preferably 5%, 4%, 3%, 2%, 1%, 0.1% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position therebetween, individually interspersed among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, by having a polypeptide having a given amino acid sequence that has at least, for example, 85%, preferably 90%, 91%, 92%, 93%, 94%, even more preferably 95%, 96%, 97%, 98%, 99% 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 at most 15, preferably at most 10, 9, 8, 7, 6, even more preferably at most 5, 4, 3, 2, or 1 amino acid alterations per 100 amino acids of the reference amino acid sequence.In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, 91%, 92%, 93%, 94%, even more preferably 95%, 96%, 97%, 98%, 99% sequence identity to a reference amino acid sequence, up to 15%, preferably up to 10%, 9%, 8%, 7%, even more preferably up to 5%, 4%, 3%, 2%, 1% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a certain number of amino acids up to 15%, preferably up to 10%, 9%, 8%, 7%, even more preferably up to 5%, 4%, 3%, 2%, 1% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. Such alterations of the reference sequence may occur at the amino-terminal or carboxyl-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, either individually interspersed among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. However, when determining sequence identity, conservative substitutions are not included as matches.

[0121] The terms "sequence identity" or "percent identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the most preferred comparison purpose (e.g., a gap can be introduced into the first amino acid or nucleic acid sequence for most preferred comparison with the second amino acid or nucleic acid sequence). Subsequently, the amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecule at that position has identity. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are the same length.

[0122] Sequence comparison can be performed over the full length of the two sequences being compared or over fragments of the two sequences. Within the scope of the present invention, it is generally and preferably to compare over the full length of the two sequences being compared. However, sequence identity can be performed over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[0123] As used herein, it will in particular be understood that the term "having at least X% sequence identity to the nucleic acid / amino acid sequence according to SEQ ID NO: Y" (or alternatively, the term "having at least X% sequence identity to the nucleic acid / amino acid sequence as set forth in / of SEQ ID NO: Y") is equivalent to the term "having at least X% sequence identity to the nucleic acid / amino acid sequence according to SEQ ID NO: Y over the length of SEQ ID NO: Y" or to the term "having at least X% sequence identity to the nucleic acid / amino acid sequence according to SEQ ID NO: Y over the full length of SEQ ID NO: Y", respectively.

[0124] Those skilled in the art will appreciate that several different computer programs can be used to determine the homology between two sequences. For example, sequence comparison and the determination of the percent identity between two sequences can be achieved using a mathematical algorithm. In one particular aspect, the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), is used to determine the percent identity between two amino acid or nucleic acid sequences using a Blosum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Those skilled in the art will appreciate that all these different parameters will produce slightly different results, but the overall percent identity of the two sequences does not change significantly when different algorithms are used.

[0125] The protein or nucleic acid sequences of the invention can further be used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0126] For the purposes of this invention, the term "suid" refers to any animal in the genus Sus (Sus). The term "suid" includes domestic pigs (Sus scrofa domesticus) and wild pigs (Sus scrofascrofa), as well as warthogs (Potamochoerus porcus), bush pigs (Potamochoerus larvatus), giant forest hogs (Hylochoerus meinertzhageni), and feral pigs. It should be understood that suids encompass both male and female animals. Semen can contain ASFV, and for this reason, the term "suid" encompasses both male and female breeding animals. Thus, the terms "suid" and "pig" encompass male animals, such as boars, and female animals, such as sows and gilts. As used herein, the term "swines" refers to suids, preferably pigs, before and during their first pregnancy. In contrast, the term "sow" as used herein refers to a porcine animal, preferably a pig, after first farrowing, with farrowing being a positive outcome of its first pregnancy. Preferably, the "porcine animal" is a pig, in particular a domestic pig.

[0127] An "immunogenic composition" or "immune composition" generally refers to a composition comprising a substance containing at least one antigen or an immunogenic portion thereof that elicits an immune response in a host, either a cellular immune response or an antibody-mediated immune response, against the composition. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more of the clinical symptoms of ASFV infection. An immunogenic composition is described as a "vaccine" when the host exhibits a protective immune response resulting in increased resistance to the novel infection and / or reduced clinical severity of the disease.

[0128] The term "antigen" as used herein is well understood in the art and includes immunogenic substances (i.e., immunogens) as well as substances that induce immune anergy or inertia (i.e., the lack of a response by the body's defense mechanisms against a foreign substance). As used herein, the term "antigen" is intended to mean a full-length protein that includes or contains an epitope, as well as peptide fragments thereof. In addition, the term "antigen coding sequence" refers to a sequence that encodes an antigen. Preferably, the antigen coding sequence is a nucleic acid sequence such as a cDNA sequence.

[0129] An “immunogenic composition” as specifically described and claimed herein contains the following substances that elicit an immune response and / or immunogenicity as described herein: one, two or more ASFV peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof, such as ASFV surface proteins and / or immunogenic fragments thereof; or one, two or more ASFV oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding ASFV peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; or a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, which contains one, two or more ASFV oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding ASFV peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof.

[0130] In the present invention, the term "immunogenic fragment" or "immunogenic portion" or "immunogenic fragment thereof" refers to fragments or truncated and / or substituted forms of ASFV peptides, polypeptides, or full-length proteins, as well as fragments or truncated and / or substituted forms of corresponding ASFV-encoding oligonucleotides or polynucleotides, all of which contain one or more epitopes and thus elicit an immune response and / or immunogenicity as described herein. In the present invention, for example, an immunogenic fragment of a full-length ASFV protein can be an ASFV polypeptide or an ASFV peptide. Depending on the length and / or properties of such ASFV peptide, such an ASFV peptide can also contain more than one epitope—thus, in the present invention, it is even possible that one or more immunogenic fragments of a given ASFV peptide exist, depending on the number of epitopes contained. Furthermore, in the present invention, for example, an immunogenic fragment of a corresponding ASFV-encoding polynucleotide can be a corresponding ASFV-encoding oligonucleotide. Depending on the length and / or nature of such ASFV oligonucleotides, the ASFV oligonucleotides may also contain more than one epitope - thus, in the process of the present invention, it is also possible that one or more immunogenic fragments of a given ASFV oligonucleotide are present - depending on the number of epitopes contained. In addition, in the process of the present invention, the ASFV oligonucleotides and / or polynucleotides themselves are immunogenic, that is, the given nucleic acid is therefore immunogenic and contains at least one epitope and thus elicits an immune response and / or immunogenicity as described herein. Alternatively, in the process of the present invention, the ASFV oligonucleotides and / or polynucleotides themselves are not immunogenic, but encode an ASFV peptide, polypeptide or full-length protein that is immunogenic and contains at least one epitope and thus elicits an immune response and / or immunogenicity as described herein.Preferably, the fragments or truncated and / or substituted forms of the ASFV peptide, polypeptide or full-length protein and the corresponding fragments or truncated and / or substituted forms of the ASFV oligonucleotide or polynucleotide encoding the ASFV contain 2 to 1000, 3 to 500, 4 to 300, 5 to 200, 6 to 180 or 7 to 150 contiguous amino acid residues relative to the length of the ASFV peptide, polypeptide or full-length protein; more preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200 or more contiguous amino acid residues. , 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more contiguous nucleotides.

[0131] Such fragments can be identified using any number of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by simultaneously synthesizing a large number of peptides corresponding to portions of the protein molecule on a solid support and reacting the peptides with antibodies while the peptides are still attached to the support. Such techniques are known and described in the art, see, for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; and Geysen et al. (1986) Molec. Immunol. 23: 709-715. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, such as by using, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols, supra. Synthetic antigens, such as multiple epitopes, flanking epitopes, and other recombinant or synthetically derived antigens, are also included within the definition. See, for example, Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 75:402-408; and Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. The teachings and contents of these references are incorporated herein by reference in their entirety.

[0132] In the present invention, the term "ASFV peptide" refers to an amino acid sequence consisting of two or more, but not more than 50 amino acid residues, more preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues, containing at least one epitope and thus eliciting an immune response and / or immunogenicity as described herein.

[0133] In the course of the present invention, the term "ASFV polypeptide" refers to an amino acid sequence consisting of more than 50 amino acid residues, containing at least one epitope and thus eliciting an immune response and / or immunogenicity as described herein.

[0134] The term "ASFV full-length protein" refers to an ASFV polypeptide, which is encoded by a polynucleotide sequence containing, more preferably consisting of: any possible open reading frame (ORF); preferably, it is encoded by a polynucleotide sequence containing, more preferably consisting of: an open reading frame (ORF) with a 5' start codon and a 3' stop codon.

[0135] In the context of the present invention, the term "ASFV oligonucleotide" refers to a nucleotide sequence of at least two, but not more than 12 nucleotides, which may contain at least one epitope and thus elicit an immune response and / or immunogenicity as described herein.

[0136] In the present invention, the term "ASFV polynucleotide" refers to a nucleotide sequence having 13 or more nucleotides that may contain at least one epitope and thus elicit an immune response and / or immunogenicity as described herein.

[0137] The term "immunizing" relates to effective immunization by administering an immunogenic composition to the porcine animal to be immunized, thereby eliciting an immune response against the antigens included in the immunogenic composition.

[0138] As used herein, the term "in need" or "of need" means that the administration or treatment is associated with an improvement or amelioration of the health status or clinical symptoms of the animal receiving the immunogenic composition of the present invention or any other positive medical effect on the health status of the animal.

[0139] As used herein, the term "vaccine" refers to a pharmaceutical composition containing at least one immunologically active component that induces an immune response in an animal and, possibly but not necessarily, one or more additional components that enhance the immunological activity of the active component. A vaccine may further contain additional components typical of pharmaceutical compositions. As a distinction, the immunologically active component of a vaccine may contain whole virus particles, either in their native form, or in the form of attenuated particles in so-called modified live virus vaccines (MLVs), or in the form of particles inactivated by appropriate methods in so-called killed virus vaccines (KVs). In another form, the immunologically active component of a vaccine may contain appropriate components of an organism (subunit vaccines), such as peptides and / or polypeptides, preferably full-length proteins, and oligonucleotides and / or polynucleotides, whereby these components are produced by disruption of intact particles or cultures containing such particles, followed by an optional purification step, to obtain the desired structure, or by a synthetic process involving appropriate manipulation using suitable systems based on, for example, bacteria, insects, mammals, or other species, with optional subsequent isolation and purification procedures, or by inducing the synthetic process in an animal in need of the vaccine by directly incorporating genes or substances using suitable pharmaceutical compositions (polynucleotide vaccination). A vaccine may contain one of the components described above or may contain more than one of the components described above simultaneously. As used within a specific aspect of the present invention, "vaccine" refers to a live virus vaccine or live virus, also known as a recombinant vaccine. In another specific aspect of the present invention, "vaccine" refers to an inactivated virus or killed virus, including virus-like particles (VLPs). Thus, a vaccine may be a subunit vaccine or a killed virus vaccine (KV) or an inactivated vaccine.

[0140] The term "DNA vaccination" or "polynucleotide vaccination" means the direct vaccination of genetic material using a suitable pharmaceutical composition.

[0141] Various physical and chemical inactivation methods are known in the art. The term "inactivation" refers to the process by which a virulent or non-virulent virus has been previously irradiated (ultraviolet (UV), X-rays, electron beams, or gamma radiation), heated, or chemically treated to inactivate or kill the virus while retaining its immunogenicity. Suitable inactivating agents include β-propiolactone, diethyleneimine or β-ethyleneimine or acetyl-ethyleneimine, glutaraldehyde, ozone, and formalin (formaldehyde).

[0142] For inactivation using formalin or formaldehyde, formaldehyde is typically mixed with water and methanol to produce formalin. The addition of methanol prevents degradation or cross-reactions during the activation process. One embodiment uses approximately 0.1% to 1% of a 37% formaldehyde solution to inactivate the virus. The key is to adjust the amount of formalin to ensure that the substance is inactivated, but not to the point where high doses cause side effects.

[0143] More specifically, in the context of a virus, the term "inactivated" means that the virus is unable to replicate in vivo or in vitro. For example, the term "inactivated" may refer to a virus that has been propagated in vitro and then inactivated using chemical or physical means, rendering it no longer able to replicate.

[0144] As used herein, the terms "inactivated," "killed virus," or "KV" are used interchangeably.

[0145] The term "live virus vaccine" refers to a vaccine that contains a living organism or a replication-competent virus or viral vector.

[0146] A "pharmaceutical composition" consists essentially of one or more ingredients capable of improving a physiological function (e.g., immune function) of an organism to which it is administered or of an organism living in or on that organism. This term includes, but is not limited to, antibiotics or antiparasitic agents, as well as other components commonly used to achieve certain other objectives, such as, but not limited to, processing characteristics, sterility, stability, feasibility of administering the composition enterally or via parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal, or other suitable routes, post-administration tolerance, or controlled-release properties. A non-limiting example of such a pharmaceutical composition, given for illustrative purposes only, can be prepared by mixing cell culture supernatant from infected cell cultures with a stabilizer (e.g., spermidine and / or bovine serum albumin (BSA)) and subsequently lyophilizing or dehydrating the mixture by other methods. Prior to vaccination, the mixture is then reconstituted in an aqueous solution (e.g., saline, phosphate-buffered saline (PBS)) or a non-aqueous solution (e.g., an oil emulsion, an aluminum-based adjuvant).

[0147] As used herein, "pharmaceutically or veterinarily acceptable carriers" include any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In certain aspects, and particularly including aspects of lyophilized immunogenic compositions, stabilizers for use in the present invention include stabilizers for lyophilization or freeze-drying.

[0148] In some embodiments, the immunogenic composition of the present invention comprises an adjuvant. As used herein, "adjuvant" may 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 emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsion may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils such as squalane or squalene; oils produced by oligomerization of olefins, specifically isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, specifically isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, in particular, optionally ethoxylated esters of sorbitan, esters of mannide (e.g., anhydrous mannitol oleate), esters of ethylene glycol, polyglycerol, propylene glycol, and esters of oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid; and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51-94 (1995), and Todd et al., Vaccine 15: 564-570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach", eds. M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0149] Another embodiment of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid crosslinked, in particular, with polyalkenyl ethers of sugars or polyols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic acid polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8 hydroxyl groups, wherein the hydrogen atoms of at least three hydroxyl groups are replaced by unsaturated aliphatic groups having at least 2 carbon atoms. Preferred groups are unsaturated aliphatic groups comprising 2 to 4 carbon atoms, such as vinyl, allyl and other olefinically unsaturated groups. The unsaturated groups themselves may contain other substituents such as methyl. Trade names include CARBOMER. The products sold by BF Goodrich, Ohio, USA are particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. Particular mention may be made of Carbopol 974P, 934P and 971P. Most preferably, the use 971P. Among the copolymers of maleic anhydride and alkenyl derivatives is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. These polymers are dissolved in water to produce an acid solution, which is preferably neutralized to physiological pH to obtain an adjuvant solution with the immunogen, immune or vaccine composition itself.

[0150] Additional suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (E. coli) (recombinant or otherwise), cholera toxin IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or endogenous cytokine release stimulators.

[0151] It is contemplated that the adjuvant may be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be added in a concentration of about 0.01% to 50%, preferably in a concentration of about 2% to 30%, more preferably in a concentration of about 5% to 25%, even more preferably in a concentration of about 7% to 22%, and most preferably in a concentration of 10% to 20% by volume of the final product.

[0152] "Diluents" may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include, among others, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, among others, albumin and alkali metal salts of ethylenediaminetetraacetic acid.

[0153] "Isolated" means altered "by the hand of man" from its natural state, that is, if it occurs in nature, it is changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated" as the term is used herein, but the same polynucleotide or polypeptide, separated from the coexisting materials of its natural state, is "isolated."

[0154] "Attenuation" means reducing the toxicity of a pathogen. In the present invention, "attenuation" is synonymous with "avirulence." In the present invention, an attenuated virus is a virus whose toxicity has been reduced so that it does not cause clinical symptoms of infection but is able to induce an immune response in the target animal, but it can also mean that the incidence or severity of clinical symptoms in animals infected with the attenuated virus is reduced compared to a "control group" of animals infected with a non-attenuated virus or pathogen and not receiving the attenuated virus. In this context, the term "reduce" or "reduced" means a reduction of at least 10%, preferably 25%, even more preferably 50%, even more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90%, and most preferably 100%, compared to a control group as defined above. Therefore, attenuated, avirulent pathogens such as the claimed attenuated viruses or bacterial vectors are suitable for use in the production of modified live virus vaccines (MLVs) or modified live immunogenic compositions.

[0155] The terms "treating and / or preventing" and "reducing or preventing clinical symptoms or disease" refer to reducing the incidence of a specific ASFV infection or reducing the severity of clinical symptoms caused by or associated with a specific ASFV infection. Therefore, the terms "treating and / or preventing" and "reducing or preventing clinical symptoms or disease" also refer to reducing the number of animals infected with a specific ASFV (= reducing the incidence of ASFV infection) or reducing the severity of clinical symptoms typically associated with or caused by an ASFV infection in a group of animals that have received an effective amount of an immunogenic composition as provided herein, compared to a group of animals that have not received such an immunogenic composition. The terms "treating and / or preventing" and "reducing or preventing clinical symptoms or disease" generally relate to administering an effective amount of an immunogenic composition of the present invention to one or more animals in need of or that may benefit from such treatment / prevention / reduction / prevention. The term "treatment" refers to the administration of an effective amount of an immunogenic composition once an animal or at least some animals have been infected with the ASFV and wherein the animals have shown some clinical symptoms caused by or associated with the ASFV infection. The terms "control" and "prevention" refer to the administration to an animal before the animal is infected with any ASFV or at least when the animal does not show, or none of the animals in a group of animals show, any clinical symptoms caused by or associated with the ASFV infection. The terms "control" and "prevention" are used interchangeably in this application.

[0156] As used herein, the term "clinical symptoms" refers to symptoms of ASFV infection in animals. Clinical infection symptoms vary depending on the ASFV strain selected. Examples of such clinical symptoms include, but are not limited to, increased thirst, increased urination, weight loss, decreased appetite, lethargy, vomiting, viremia, fever, and environmental shedding of toxins. However, clinical symptoms also include, but are not limited to, those directly observable in live animals.

[0157] Preferably, the clinical symptoms that are reduced in incidence or severity in treated animals compared to animals that are not treated or treated with an immunogenic composition available before the present invention but are subsequently infected with a specific ASFV are African swine fever, acute African swine fever, chronic African swine fever, mortality, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleed, abortion, leukopenia, thrombocytopenia.

[0158] Herein, "effective dose" means but is not limited to the amount of an antigen that induces or is capable of eliciting an immune response that produces a reduction in clinical symptoms in an animal to which the antigen is administered.

[0159] As used herein, the term "effective amount" in the context of a composition means an amount of an immunogenic composition that is capable of inducing an immune response in an animal that reduces the incidence or severity of an infection or disease event. This effective amount is capable of reducing the incidence of a specific ASFV infection in a porcine animal or reducing the severity of clinical symptoms of a specific ASFV infection. Specifically, an effective amount refers to colony forming units (CFU) per dose. Alternatively, in the context of a therapy, the term "effective amount" refers to an amount of a therapy sufficient to reduce or ameliorate the severity or duration of a disease or condition or one or more symptoms thereof, prevent the progression of a disease or condition, cause regression of a disease or condition, prevent the recurrence, manifestation, onset or progression of one or more symptoms associated with a disease or condition, or enhance or improve the prophylactic or therapeutic effects of another therapy or therapeutic agent.

[0160] "Immune response" or "immunological response" or "immunogenicity" means, but is not limited to, the development of a cellular immune response and / or an antibody-mediated immune response to the (immunogenic) composition or vaccine of interest. Generally, the immune response or immunogenicity 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 that are specific for one or more antigens included in the composition or vaccine of interest. Preferably, the host will exhibit a therapeutic or protective immune (memory) response such that resistance to the new infection is enhanced and / or the clinical severity of the disease is reduced. This protection will be demonstrated by a reduction in the number of symptoms, a reduction in the severity of symptoms, or an absence of one or more symptoms associated with infection with a pathogen, a delay in the onset of viremia, a reduction in viral persistence, a reduction in overall viral load, and / or a reduction in viral secretion.

[0161] In the context of the present invention, an "immune response" or "immunogenicity" is preferably indicated / characterized by an induced IFN-γ response in a porcine IFN-γ ELISpot assay, more preferably in a porcine IFN-γ ELISpot assay as described in Example 1. An exemplary porcine IFN-γ ELISpot assay that can be used according to the present invention is as follows: IFNγ responses are assessed by ELISpot assay using purified mouse anti-porcine IFNγ clone P2G10 as the capture antibody and biotinylated mouse anti-porcine IFNγ antibody P2C11 as the detection antibody, following previously reported methods (Lacasta et al., 2014). Briefly, 96-well plates were coated overnight at 4°C with 5 μg / ml of capture antibody in carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with PBS and blocked with complete RPMI with 10% FBS for 1 hour at 37°C. 5×10 cells were plated in the presence of the corresponding stimulant. 5PBMC / well, final volume 200μl. Add peptides and / or polypeptides, preferably full-length proteins, as stimuli at a final concentration of 4μg / ml. Use RPMI and 10μg / ml phytohemagglutinin-M as negative and positive controls, respectively. When using live attenuated virus BA71ΔCD2 as a stimulus, add 10 5 PFU. After overnight incubation at 37°C, 5% CO2, the cells were washed with PBS 0.05% Tween 20, and IFNγ was detected using 0.5 μg / ml biotinylated anti-porcine IFNγ antibody for 1 hour at 37°C. After washing, the ELISpot was developed by adding 50 μl of insoluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate and stopped by washing with water.

[0162] "Disease prevention," "protective immunity," "functional immunity," "reduction of clinical signs / symptoms," "neutralizing antibody induction / production and / or seroconversion," and similar phrases mean that a partial or complete response to a disease or condition is generated by administering one or more immunogenic compositions or vaccines or pharmaceutical compositions of the present invention, or a combination thereof, resulting in less adverse effects than would be expected in an unvaccinated animal that had been exposed to the disease or infection. That is, the severity of the adverse effects of the infection is reduced in the vaccinated animal. The infection in the vaccinated animal may be alleviated, slowed, or potentially completely prevented. Herein, where complete prevention of infection is intended, complete prevention is specifically stated. If complete prevention is not stated, the term includes partial prevention. A "protective immune response" or "protective immunity" will be demonstrated by a reduction or absence of clinical signs / symptoms normally exhibited by an infected host, a faster recovery time and / or a shorter duration of infection, or a reduction in the titer of the pathogen in the tissues, body fluids, or excreta of the infected host.

[0163] As used herein, "reducing the incidence and / or severity of clinical signs" or "reducing clinical symptoms" or "reducing or preventing clinical symptoms or disease" means, but is not limited to, reducing the number of infected animals in a group, reducing or eliminating the number of animals displaying clinical signs of infection, or reducing the severity of any clinical signs present in one or more animals compared to a wild-type infection. For example, this would refer to any reduction in pathogen load, pathogen shedding, pathogen transmission, or a reduction in any clinical signs symptomatic of ASFV infection. Preferably, such clinical signs / symptoms are reduced by at least 10% in one or more animals receiving the therapeutic composition of the invention compared to infected animals that did not receive the composition. More preferably, clinical signs / symptoms are reduced by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50% in animals receiving the composition of the invention.

[0164] The term "increased protection" herein means, but is not limited to, a statistically significant reduction in one or more clinical signs or symptoms associated with infection by an infectious agent in a vaccinated group of animals compared to a control group of unvaccinated animals. The term "statistically significant reduction in clinical symptoms" means, but is not limited to, that after challenge with an infectious agent, the frequency of at least one clinical symptom in a vaccinated group of animals is at least 10%, preferably 20%, more preferably 30%, even more preferably 50%, and even more preferably 70% lower than in an unvaccinated control group.

[0165] The term "pathogen" is well known to those skilled in the art. The term "pathogen" includes bacteria and viruses. In the present invention, the term "pathogen that infects porcine animals" is preferably ASFV.

[0166] "Long-lasting protection" shall mean "improved efficacy" that lasts for at least 3 weeks, but more preferably at least 3 months, and even more preferably at least 6 months.

[0167] The term "shedding" refers to secretions such as nasal mucus, and further refers to aerosols produced by coughing or sneezing. Therefore, shedding can be measured by testing the viral titer in a nasal swab or the viral titer in the lungs. The term "shedding" further encompasses the transfer of the virus to susceptible animals (i.e., sentinels). Methods for measuring shedding are within the common knowledge of those skilled in the art.

[0168] "Safety" refers to the absence of adverse consequences in vaccinated animals following vaccination, including but not limited to, possible restoration of virulence of the vaccine-based pathogen, clinically significant side effects such as persistent systemic disease, or unacceptable inflammation at the site of vaccine administration.

[0169] As used herein, the term "vaccination" or "vaccinating" or variations thereof means, but is not limited to, a process comprising administering an immunogenic composition of the present invention that, when administered to an animal, directly or indirectly elicits or is capable of eliciting an immune response in the animal.

[0170] In the context of the present invention, "mortality" refers to death caused by infection and includes situations where the infection is so severe that the animal must be euthanized to prevent suffering and provide for the humane end of its life.

[0171] The formulations of the present invention contain an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable vehicle. Vaccines contain an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable vehicle. The formulation should be suitable for the mode of administration.

[0172] Optionally, the immunogenic composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. The immunogenic composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0173] Preferred routes of administration include, but are not limited to, intranasal, oral, intradermal, subcutaneous, and intramuscular. It is desirable to administer the drug in drinking water, and most preferably a single dose is administered. Those skilled in the art will appreciate that the compositions of the present invention may also be administered in one, two, or more doses, and by other routes of administration. For example, such other routes include subcutaneous, intradermal, and intraperitoneal, and depending on the desired duration and effectiveness of treatment, the compositions of the present invention may be administered once or several times, or intermittently, for example, on a daily basis for several days, weeks, or months, and in different doses, such as about 1×10 3 to 1×10 9 In a specific aspect of the invention, the dose is about 1×10 4 to 1×10 8 TCID 50 .

[0174] The term "sample" refers to a body fluid sample, a separated cell sample, or a tissue or organ sample. Body fluid samples can be obtained by well-known techniques and preferably include blood, plasma, serum, or urine samples, more preferably blood, plasma, or serum samples. Tissue or organ samples can be obtained from any tissue or organ, for example, by biopsy. Separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting.

[0175] The term "obtaining" may include separation and / or purification steps known to those skilled in the art, preferably using precipitation, columns, etc.

[0176] The terms "immunoassay" and "genomic analysis test" are the basis for distinguishing animals vaccinated with the immunogenic composition of the present invention from animals infected with naturally occurring (disease-associated) ASFV. Examples of immunoassays include any enzyme immunoassay or immunochemical detection method such as ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), sandwich enzyme immunoassay, fluorescent antibody test (FAT), electrochemiluminescent sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluoride immunoassay (DELFIA) or solid phase immunoassay, immunofluorescence test (IFT), immunohistological staining, immunoblot analysis, or any other suitable method available to those skilled in the art. Depending on the assay used, the antigen or antibody may be labeled with an enzyme, a fluorophore, or a radioisotope. See, for example, Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, NY (1994); and Frye et al., Oncogen 4: 1153-1157, 1987.

[0177] The term "genomic profiling test" refers to genomic profiling methods based on polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR (r-PCR) or real-time reverse transcription PCR (rRT-PCR), Templex-PCR, nucleic acid sequence-based amplification (NASBA), and isothermal amplification methods using polymerases and specific oligonucleotides as primers. These amplification methods are well known in the art.

[0178] Hybridization reactions can be performed under different "stringency" conditions. Conditions that increase the stringency of hybridization reactions are well known. See, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al. 1989). Examples of relevant conditions (for increasing stringency) include: incubation temperatures of 25°C, 37°C, 50°C, and 68°C; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (wherein SSC is 0.15M NaCl and 15mM citrate buffer) and their equivalent concentrations using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times of 5 minutes to 24 hours; 1, 2, or more wash steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water.

[0179] The term "epitope" refers to a site on an antigen, such as an ASFV peptide, polypeptide, or full-length protein, or ASFV oligonucleotide or polynucleotide of the present invention, that is the target of a specific B- and / or T-cell response. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." Antibodies that recognize the same epitope can be identified in a simple immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to the target antigen.

[0180] Sequence Listing

[0181] This application includes a sequence listing. The sequence listing contains the following sequences that are amino acid sequences (N-terminal to C-terminal direction) or DNA sequences (5' to 3' direction):

[0182] SEQ ID NO: 1 A118R peptide Georgia 2007 / 1, 23-43, 21

[0183] SEQ ID NO: 2 A118R peptide Georgia 2007 / 1, 33-43, 11

[0184] SEQ ID NO: 3 A118R peptide Georgia 2007 / 1, 41-50, 10

[0185] SEQ ID NO:4 A118R protein Georgia 2007 / 1

[0186] SEQ ID NO:5 A137R peptide BA71

[0187] SEQ ID NO:6 A137R protein BA71

[0188] SEQ ID NO:7 A137R protein Georgia 2007 / 1

[0189] SEQ ID NO:8 A151R peptide BA71

[0190] SEQ ID NO:9 A151R peptide Georgia 2007 / 1, 40-55, 16

[0191] SEQ ID NO: 10 A151R peptide Georgia 2007 / 1, 43-52, 10

[0192] SEQ ID NO: 11 A151R peptide Georgia 2007 / 1, 48-55, 8

[0193] SEQ ID NO:12 A151R protein BA71

[0194] SEQ ID NO:13 A151R protein Georgia 2007 / 1

[0195] SEQ ID NO: 14 A179L peptide BA71

[0196] SEQ ID NO:15 A179L protein BA71

[0197] SEQ ID NO:16 A179L protein Georgia 2007 / 1

[0198] SEQ ID NO: 17 A238L peptide BA71

[0199] SEQ ID NO: 18 A238L peptide BA71

[0200] SEQ ID NO: 19 A238L peptide Georgia 2007 / 1

[0201] SEQ ID NO:20 A238L peptide Georgia 2007 / 1

[0202] SEQ ID NO:21 A238L protein BA71

[0203] SEQ ID NO:22 A238L protein BA71

[0204] SEQ ID NO:23 A238L protein Georgia 2007 / 1

[0205] SEQ ID NO:24 A238L protein Georgia 2007 / 1

[0206] SEQ ID NO:25 A240L peptide Georgia 2007 / 1

[0207] SEQ ID NO:26 A240L peptide Georgia 2007 / 1

[0208] SEQ ID NO:27 A240L protein Georgia 2007 / 1

[0209] SEQ ID NO:28 A859L peptide Georgia 2007 / 1, 572-580, 9

[0210] SEQ ID NO:29 A859L peptide Georgia 2007 / 1, 626-636, 11

[0211] SEQ ID NO:30 A859L protein Georgia 2007 / 1

[0212] SEQ ID NO:31 B117L peptide BA71

[0213] SEQ ID NO:32 B117L protein BA71

[0214] SEQ ID NO:33 B117L protein Georgia 2007 / 1

[0215] SEQ ID NO:34 B119L peptide Georgia 2007 / 1, 68-75, 8

[0216] SEQ ID NO:35 B119L protein Georgia 2007 / 1

[0217] SEQ ID NO:36 B125R peptide BA71

[0218] SEQ ID NO:37 B125R peptide BA71

[0219] SEQ ID NO:38 B125R protein BA71

[0220] SEQ ID NO:39 B125R protein Georgia 2007 / 1

[0221] SEQ ID NO:40 B169L peptide Georgia 2007 / 1, 26-34, 9

[0222] SEQ ID NO:41 B169L protein Georgia 2007 / 1

[0223] SEQ ID NO:42 B175L peptide Georgia 2007 / 1, 69-78, 10

[0224] SEQ ID NO:43 B175L protein Georgia 2007 / 1

[0225] SEQ ID NO:44 B318L peptide Georgia 2007 / 1, 155-162, 8

[0226] SEQ ID NO:45 B318L protein Georgia 2007 / 1

[0227] SEQ ID NO:46 B385R peptide Georgia 2007 / 1, 180-188, 9

[0228] SEQ ID NO:47 B385R protein Georgia 2007 / 1

[0229] SEQ ID NO:48 B475L peptide BA71

[0230] SEQ ID NO:49 B475L peptide BA71

[0231] SEQ ID NO:50 B475L peptide BA71

[0232] SEQ ID NO:51 B475L peptide BA71

[0233] SEQ ID NO:52 B475L peptide BA71

[0234] SEQ ID NO:53 B475L peptide BA71

[0235] SEQ ID NO:54 B475L peptide Georgia 2007 / 1

[0236] SEQ ID NO:55 B475L peptide Georgia 2007 / 1

[0237] SEQ ID NO:56 B475L peptide Georgia 2007 / 1

[0238] SEQ ID NO:57 B475L peptide Georgia 2007 / 1

[0239] SEQ ID NO:58 B475L peptide Georgia 2007 / 1

[0240] SEQ ID NO:59 B475L peptide Georgia 2007 / 1

[0241] SEQ ID NO:60 B475L peptide Georgia 2007 / 1, 10-18, 9

[0242] SEQ ID NO:61 B475L peptide Georgia 2007 / 1, 14-24, 11

[0243] SEQ ID NO:62 B475L peptide Georgia 2007 / 1, 14-28, 15

[0244] SEQ ID NO:63 B475L peptide Georgia 2007 / 1, 18-28, 11

[0245] SEQ ID NO:64 B475L peptide Georgia 2007 / 1, 62-70, 9

[0246] SEQ ID NO:65 B475L protein BA71

[0247] SEQ ID NO:66 B475L protein Georgia 2007 / 1

[0248] SEQ ID NO:67 B602L peptide BA71

[0249] SEQ ID NO:68 B602L peptide BA71

[0250] SEQ ID NO:69 B602L peptide BA71

[0251] SEQ ID NO:70 B602L peptide Georgia 2007 / 1, 54-77, 24

[0252] SEQ ID NO:71 B602L peptide Georgia 2007 / 1, 61-69, 9

[0253] SEQ ID NO:72 B602L peptide Georgia 2007 / 1, 61-71, 11

[0254] SEQ ID NO:73 B602L peptide Georgia 2007 / 1, 73-81, 9

[0255] SEQ ID NO:74 B602L protein BA71

[0256] SEQ ID NO:75 B602L protein Georgia 2007 / 1

[0257] SEQ ID NO:76 B646L peptide BA71

[0258] SEQ ID NO:77 B646L peptide BA71

[0259] SEQ ID NO:78 B646L peptide BA71

[0260] SEQ ID NO:79 B646L peptide BA71

[0261] SEQ ID NO:80 B646L peptide Georgia 2007 / 1, 455-465, 11

[0262] SEQ ID NO:81 B646L peptide Georgia 2007 / 1, 457-465, 9

[0263] SEQ ID NO:82 B646L protein BA71

[0264] SEQ ID NO:83 B646L protein Georgia 2007 / 1

[0265] SEQ ID NO:84 B962L peptide Georgia 2007 / 1, 599-606, 8

[0266] SEQ ID NO:85 B962L protein Georgia 2007 / 1

[0267] SEQ ID NO:86 BA71V-A104R protein BA71

[0268] SEQ ID NO:87 BA71V-A104R protein Georgia 2007 / 1

[0269] SEQ ID NO:88 BA71V-A118R protein BA71

[0270] SEQ ID NO:89 BA71V-A118R protein Georgia 2007 / 1

[0271] SEQ ID NO:90 BA71V-A137R (p11.5) protein BA71

[0272] SEQ ID NO:91 BA71V-A137R (p11.5) protein Georgia 2007 / 1

[0273] SEQ ID NO:92 BA71V-A179L(5HL)Bcl2 protein BA71

[0274] SEQ ID NO:93 BA71V-A179L (5HL) Bcl2 protein Georgia 2007 / 1

[0275] SEQ ID NO:94 BA71V-A224L (4CL) protein BA71

[0276] SEQ ID NO:95 BA71V-A224L (4CL) protein Georgia 2007 / 1

[0277] SEQ ID NO:96 BA71V-A238L (5EL) protein BA71

[0278] SEQ ID NO:97 BA71V-A238L (5EL) protein Georgia 2007 / 1

[0279] SEQ ID NO:98 BA71V-A859L protein BA71

[0280] SEQ ID NO:99 BA71V-A859L protein Georgia 2007 / 1

[0281] SEQ ID NO: 100 BA71V-B175L protein BA71

[0282] SEQ ID NO: 101 BA71V-B175L protein Georgia 2007 / 1

[0283] SEQ ID NO: 102 BA71V-B263R protein BA71

[0284] SEQ ID NO: 103 BA71V-B263R protein Georgia 2007 / 1

[0285] SEQ ID NO: 104 BA71V-B407L protein BA71

[0286] SEQ ID NO: 105 BA71V-B407L protein Georgia 2007 / 1

[0287] SEQ ID NO: 106 BA71V-B438L (p49) protein BA71

[0288] SEQ ID NO: 107 BA71V-B438L (p49) protein Georgia 2007 / 1

[0289] SEQ ID NO: 108 BA71V-C62L protein BA71

[0290] SEQ ID NO: 109 BA71V-C62L protein Georgia 2007 / 1

[0291] SEQ ID NO: 110 BA71V-D129L protein BA71

[0292] SEQ ID NO: 111 BA71V-D129L protein Georgia 2007 / 1

[0293] SEQ ID NO: 112 BA71V-D250R (g5R) protein BA71

[0294] SEQ ID NO: 113 BA71V-D250R (g5R) protein Georgia 2007 / 1

[0295] SEQ ID NO: 114 BA71V-D345L (i3L, i4L) protein BA71

[0296] SEQ ID NO:115 BA71V-D345L(i3L,i4L) protein Georgia2007 / 1

[0297] SEQ ID NO: 116 BA71V-D79L (g7L) protein BA71

[0298] SEQ ID NO: 117 BA71V-D79L (g7L) protein Georgia 2007 / 1

[0299] SEQ ID NO: 118 BA71V-DP96R protein BA71

[0300] SEQ ID NO: 119 BA71V-DP96R protein Georgia 2007 / 1

[0301] SEQ ID NO: 120 BA71V-E111R (k6R) protein BA71

[0302] SEQ ID NO: 121 BA71V-E111R (k6R) protein Georgia 2007 / 1

[0303] SEQ ID NO: 122 BA71V-E120R (p14.5) protein BA71

[0304] SEQ ID NO: 123 BA71V-E120R (p14.5) protein Georgia 2007 / 1

[0305] SEQ ID NO: 124 BA71V-E165R (k1R) protein BA71

[0306] SEQ ID NO: 125 BA71V-E165R (k1R) protein Georgia 2007 / 1

[0307] SEQ ID NO: 126 BA71V-E183L (p54, j13L) protein BA71

[0308] SEQ ID NO: 127 BA71V-E183L (p54, j13L) protein Georgia 2007 / 1

[0309] SEQ ID NO: 128 BA71V-E184L (j12L) protein BA71

[0310] SEQ ID NO: 129 BA71V-E184L (j12L) protein Georgia 2007 / 1

[0311] SEQ ID NO: 130 BA71V-E199L (j18L) protein BA71

[0312] SEQ ID NO: 131 BA71V-E199L (j18L) protein Georgia 2007 / 1

[0313] SEQ ID NO: 132 BA71V-E248R (k2R) protein BA71

[0314] SEQ ID NO: 133 BA71V-E248R (k2R) protein Georgia 2007 / 1

[0315] SEQ ID NO: 134 BA71V-E296R (k4R) protein BA71

[0316] SEQ ID NO: 135 BA71V-E296R (k4R) protein Georgia 2007 / 1

[0317] SEQ ID NO: 136 BA71V-E301R (j15R) protein BA71

[0318] SEQ ID NO: 137 BA71V-E301R (j15R) protein Georgia 2007 / 1

[0319] SEQ ID NO: 138 BA71V-EP152R protein BA71

[0320] SEQ ID NO: 139 BA71V-EP152R protein Georgia 2007 / 1

[0321] SEQ ID NO: 140 BA71V-EP364R protein BA71

[0322] SEQ ID NO: 141 BA71V-EP364R protein Georgia 2007 / 1

[0323] SEQ ID NO: 142 BA71V-F165R protein BA71

[0324] SEQ ID NO: 143 BA71V-F165R protein Georgia 2007 / 1

[0325] SEQ ID NO: 144 BA71V-F317L protein BA71

[0326] SEQ ID NO: 145 BA71V-F317L protein Georgia 2007 / 1

[0327] SEQ ID NO: 146 BA71V-H124R protein BA71

[0328] SEQ ID NO: 147 BA71V-H124R protein Georgia 2007 / 1

[0329] SEQ ID NO:148 BA71V-H171R(j2R) protein BA7

[0330] SEQ ID NO: 149 BA71V-H171R (j2R) protein Georgia 2007 / 1

[0331] SEQ ID NO: 150 BA71V-H359L (j1L) protein BA71

[0332] SEQ ID NO: 151 BA71V-H359L (j1L) protein Georgia 2007 / 1

[0333] SEQ ID NO: 152 BA71V-I177L (k14L) protein BA71

[0334] SEQ ID NO: 153 BA71V-I177L (k14L) protein Georgia 2007 / 1

[0335] SEQ ID NO: 154 BA71V-I196L (k15L) protein BA71

[0336] SEQ ID NO: 155 BA71V-I196L (k15L) protein Georgia 2007 / 1

[0337] SEQ ID NO: 156 BA71V-I215L (k13L) protein BA71

[0338] SEQ ID NO: 157 BA71V-I215L (k13L) protein Georgia 2007 / 1

[0339] SEQ ID NO: 158 BA71V-I267L (k7L) protein BA71

[0340] SEQ ID NO: 159 BA71V-I267L (k7L) protein Georgia 2007 /

[0341] SEQ ID NO: 160 BA71V-K205R protein BA71

[0342] SEQ ID NO: 161 BA71V-K205R protein Georgia 2007 / 1

[0343] SEQ ID NO: 162 BA71V-K78R (p10) protein BA71

[0344] SEQ ID NO: 163 BA71V-K78R (p10) protein Georgia 2007 / 1

[0345] SEQ ID NO: 164 BA71V-KP177R protein BA71

[0346] SEQ ID NO: 165 BA71V-KP177R protein Georgia 2007 / 1

[0347] SEQ ID NO: 166 BA71V-O174L protein BA71

[0348] SEQ ID NO: 167 BA71V-O174L protein Georgia 2007 / 1

[0349] SEQ ID NO: 168 BA71V-S183 (i5L) protein BA71

[0350] SEQ ID NO: 169 BA71V-S183 (i5L) protein Georgia 2007 / 1

[0351] SEQ ID NO: 170 BA71V-S273R (i6R) protein BA71

[0352] SEQ ID NO: 171 BA71V-S273R (i6R) protein Georgia 2007 / 1

[0353] SEQ ID NO: 172 BA71V-X69R protein BA71

[0354] SEQ ID NO: 173 BA71V-X69R protein Georgia 2007 / 1

[0355] SEQ ID NO: 174 C129R peptide BA71

[0356] SEQ ID NO: 175 C129R peptide BA71

[0357] SEQ ID NO: 176 C129R protein BA71

[0358] SEQ ID NO: 177 C129R protein Georgia 2007 / 1

[0359] SEQ ID NO: 178 C147L peptide Georgia 2007 / 1, 10-18, 9

[0360] SEQ ID NO: 179 C147L protein Georgia 2007 / 1

[0361] SEQ ID NO: 180 C257L peptide BA71

[0362] SEQ ID NO: 181 C257L protein BA71

[0363] SEQ ID NO: 182 C257L protein Georgia 2007 / 1

[0364] SEQ ID NO: 183 C315R peptide BA71

[0365] SEQ ID NO: 184 C315R peptide Georgia 2007 / 1, 257-267, 11

[0366] SEQ ID NO: 185 C315R peptide Georgia 2007 / 1, 290-299, 10

[0367] SEQ ID NO: 186 C315R protein BA71

[0368] SEQ ID NO: 187 C315R protein Georgia 2007 / 1

[0369] SEQ ID NO: 188 C475L peptide BA71

[0370] SEQ ID NO: 189 C475L peptide BA71

[0371] SEQ ID NO: 190 C475L peptide BA71

[0372] SEQ ID NO: 191 C475L peptide BA71

[0373] SEQ ID NO: 192 C475L peptide Georgia 2007 / 1, 115-123, 9

[0374] SEQ ID NO: 193 C475L peptide Georgia 2007 / 1, 127-137, 11

[0375] SEQ ID NO: 194 C475L peptide Georgia 2007 / 1, 130-137, 8

[0376] SEQ ID NO: 195 C475L peptide Georgia 2007 / 1, 207-217, 11

[0377] SEQ ID NO: 196 C475L peptide Georgia 2007 / 1, 207-221, 15

[0378] SEQ ID NO: 197 C475L peptide Georgia 2007 / 1, 210-217, 8

[0379] SEQ ID NO: 198 C475L peptide Georgia 2007 / 1, 213-221, 9

[0380] SEQ ID NO: 199 C475L peptide Georgia 2007 / 1, 438-445, 8

[0381] SEQ ID NO:200 C475L protein BA71

[0382] SEQ ID NO:201 C475L protein Georgia 2007 / 1

[0383] SEQ ID NO: 202 C62L peptide Georgia 2007 / 1, 33-51, 19

[0384] SEQ ID NO: 203 C62L peptide Georgia 2007 / 1, 41-51, 11

[0385] SEQ ID NO:204 C62L protein Georgia 2007 / 1

[0386] SEQ ID NO:205 C62L protein Georgia 2007 / 1

[0387] SEQ ID NO: 206 C717R peptide Georgia 2007 / 1, 104-116, 13

[0388] SEQ ID NO: 207 C717R peptide Georgia 2007 / 1, 356-363, 8

[0389] SEQ ID NO: 208 C717R peptide Georgia 2007 / 1, 356-366, 11

[0390] SEQ ID NO:209 C717R peptide Georgia 2007 / 1, 388-407, 20

[0391] SEQ ID NO:210 C717R peptide Georgia 2007 / 1, 394-404, 11

[0392] SEQ ID NO: 211 C717R peptide Georgia 2007 / 1, 425-435, 11

[0393] SEQ ID NO: 212 C717R peptide Georgia 2007 / 1, 47-62, 16

[0394] SEQ ID NO: 213 C717R peptide Georgia 2007 / 1, 495-505, 11

[0395] SEQ ID NO: 214 C717R peptide Georgia 2007 / 1, 543-553, 11

[0396] SEQ ID NO:215 C717R peptide Georgia 2007 / 1, 543-563, 21

[0397] SEQ ID NO:216 C717R peptide Georgia 2007 / 1, 546-553, 8

[0398] SEQ ID NO:217 C717R protein Georgia 2007 / 1

[0399] SEQ ID NO:218 C84L peptide Georgia 2007 / 1, 42-49, 8

[0400] SEQ ID NO:219 C84L protein Georgia 2007 / 1

[0401] SEQ ID NO: 220 C962R peptide Georgia 2007 / 1, 327-335, 9

[0402] SEQ ID NO: 221 C962R peptide Georgia 2007 / 1, 398-407, 10

[0403] SEQ ID NO: 222 C962R peptide Georgia 2007 / 1, 398-417, 20

[0404] SEQ ID NO: 223 C962R peptide Georgia 2007 / 1, 400-409, 10

[0405] SEQ ID NO: 224 C962R peptide Georgia 2007 / 1, 407-416, 10

[0406] SEQ ID NO: 225 C962R peptide Georgia 2007 / 1, 536-544, 9

[0407] SEQ ID NO: 226 C962R peptide Georgia 2007 / 1, 726-742, 17

[0408] SEQ ID NO: 227 C962R peptide Georgia 2007 / 1, 730-738, 9

[0409] SEQ ID NO:228 C962R protein Georgia 2007 / 1

[0410] SEQ ID NO: 229 CP123L peptide BA71

[0411] SEQ ID NO: 230 CP123L peptide BA71

[0412] SEQ ID NO:231 CP123L protein BA71

[0413] SEQ ID NO:232 CP123L protein Georgia 2007 / 1

[0414] SEQ ID NO: 233 CP204L peptide Georgia 2007 / 1, 23-33, 11

[0415] SEQ ID NO:234 ​​CP204L protein Georgia 2007 / 1

[0416] SEQ ID NO:235 CP2475L peptide BA71

[0417] SEQ ID NO: 236 CP2475L peptide BA71

[0418] SEQ ID NO: 237 CP2475L peptide BA71

[0419] SEQ ID NO: 238 CP2475L peptide BA71

[0420] SEQ ID NO: 239 CP2475L peptide BA71

[0421] SEQ ID NO: 240 CP2475L peptide BA71

[0422] SEQ ID NO: 241 CP2475L peptide BA71

[0423] SEQ ID NO: 242 CP2475L peptide BA71

[0424] SEQ ID NO: 243 CP2475L peptide BA71

[0425] SEQ ID NO: 244 CP2475L peptide Georgia 2007 / 1, 1540-1562, 23

[0426] SEQ ID NO: 245 CP2475L peptide Georgia 2007 / 1, 1547-1557, 11

[0427] SEQ ID NO: 246 CP2475L peptide Georgia 2007 / 1, 1795-1807, 13

[0428] SEQ ID NO: 247 CP2475L peptide Georgia 2007 / 1, 1889-1898, 10

[0429] SEQ ID NO: 248 CP2475L peptide Georgia 2007 / 1, 2019-2029, 11

[0430] SEQ ID NO: 249 CP2475L peptide Georgia 2007 / 1, 214-221, 8

[0431] SEQ ID NO: 250 CP2475L peptide Georgia 2007 / 1, 2226-2234, 9

[0432] SEQ ID NO: 251 CP2475L peptide Georgia 2007 / 1, 538-559, 22

[0433] SEQ ID NO: 252 CP2475L peptide Georgia 2007 / 1, 543-551, 9

[0434] SEQ ID NO: 253 CP2475L peptide Georgia 2007 / 1, 544-554, 11

[0435] SEQ ID NO: 254 CP2475L peptide Georgia 2007 / 1, 549-559, 11

[0436] SEQ ID NO: 255 CP2475L peptide Georgia 2007 / 1, 976-985, 10

[0437] SEQ ID NO:256 CP2475L protein BA71

[0438] SEQ ID NO:257 CP2475L protein Georgia 2007 / 1

[0439] SEQ ID NO:258 CP2475L_P150 peptide Georgia 2007 / 1

[0440] SEQ ID NO:259 CP2475L_P150 peptide Georgia 2007 / 1

[0441] SEQ ID NO:260 CP2475L_P150 peptide Georgia 2007 / 1

[0442] SEQ ID NO:261 CP2475L_P37 peptide Georgia 2007 / 1

[0443] SEQ ID NO:262 CP2475L_P37 peptide Georgia 2007 / 1

[0444] SEQ ID NO:263 CP2475L_P37 peptide Georgia 2007 / 1

[0445] SEQ ID NO:264 CP2475L_P37 peptide Georgia 2007 / 1

[0446] SEQ ID NO:265 CP2475L_P37 peptide Georgia 2007 / 1

[0447] SEQ ID NO:266 CP2475L_P37 peptide Georgia 2007 / 1

[0448] SEQ ID NO: 267 CP312R peptide BA71

[0449] SEQ ID NO:268 CP312R peptide Georgia 2007 / 1

[0450] SEQ ID NO:269 CP312R peptide Georgia 2007 / 1

[0451] SEQ ID NO:270 CP312R peptide Georgia 2007 / 1

[0452] SEQ ID NO:271 CP312R peptide Georgia 2007 / 1

[0453] SEQ ID NO: 272 CP312R protein BA71

[0454] SEQ ID NO:273 CP312R protein Georgia 2007 / 1

[0455] SEQ ID NO:274 CP312R protein Georgia 2007 / 1

[0456] SEQ ID NO: 275 CP530R peptide Georgia 2007 / 1, 225-235, 11

[0457] SEQ ID NO: 276 CP530R peptide Georgia 2007 / 1, 232-241, 10

[0458] SEQ ID NO: 277 CP530R peptide Georgia 2007 / 1, 358-365, 8

[0459] SEQ ID NO:278 CP530R protein Georgia 2007 / 1

[0460] SEQ ID NO: 279 CP80R peptide Georgia 2007 / 1, 67-76, 10

[0461] SEQ ID NO:280 CP80R protein Georgia 2007 / 1

[0462] SEQ ID NO:281 D1133L peptide BA71

[0463] SEQ ID NO:282 D1133L peptide BA71

[0464] SEQ ID NO: 283 D1133L peptide BA71

[0465] SEQ ID NO:284 D1133L peptide BA71

[0466] SEQ ID NO:285 D1133L peptide BA71

[0467] SEQ ID NO:286 D1133L peptide BA71

[0468] SEQ ID NO:287 D1133L peptide Georgia 2007 / 1

[0469] SEQ ID NO:288 D1133L peptide Georgia 2007 / 1

[0470] SEQ ID NO: 289 D1133L peptide Georgia 2007 / 1, 1000-1008, 9

[0471] SEQ ID NO:290 D1133L peptide Georgia 2007 / 1, 1111-1120, 10

[0472] SEQ ID NO: 291 D1133L peptide Georgia 2007 / 1, 114-124, 11

[0473] SEQ ID NO: 292 D1133L peptide Georgia 2007 / 1, 756-766, 11

[0474] SEQ ID NO: 293 D1133L peptide Georgia 2007 / 1, 782-790, 9

[0475] SEQ ID NO: 294 D1133L peptide Georgia 2007 / 1, 819-827, 9

[0476] SEQ ID NO:295 D1133L protein BA71

[0477] SEQ ID NO:296 D1133L protein BA71

[0478] SEQ ID NO:297 D1133L protein Georgia 2007 / 1

[0479] SEQ ID NO:298 D1133L protein Georgia 2007 / 1

[0480] SEQ ID NO:299 D133L peptide Georgia 2007 / 1

[0481] SEQ ID NO:300 D133L peptide Georgia 2007 / 1

[0482] SEQ ID NO:301 D133L peptide Georgia 2007 / 1

[0483] SEQ ID NO:302 D133L peptide Georgia 2007 / 1

[0484] SEQ ID NO:303 D205R peptide BA71

[0485] SEQ ID NO:304 D205R peptide BA71

[0486] SEQ ID NO:305 D205R peptide BA71

[0487] SEQ ID NO:306 D205R peptide Georgia 2007 / 1, 1-19, 19

[0488] SEQ ID NO:307 D205R peptide Georgia 2007 / 1, 2-11, 10

[0489] SEQ ID NO:308 D205R peptide Georgia 2007 / 1, 5-13, 9

[0490] SEQ ID NO:309 D205R peptide Georgia 2007 / 1, 7-17, 11

[0491] SEQ ID NO:310 D205R peptide Georgia 2007 / 1, 9-16, 8

[0492] SEQ ID NO:311 D205R protein BA71

[0493] SEQ ID NO:312 D205R protein Georgia 2007 / 1

[0494] SEQ ID NO: 313 D250R peptide BA71

[0495] SEQ ID NO:314 D250R peptide Georgia 2007 / 1, 168-178, 11

[0496] SEQ ID NO:315 D250R peptide Georgia 2007 / 1, 176-184, 9

[0497] SEQ ID NO:316 D250R peptide Georgia 2007 / 1, 32-48, 17

[0498] SEQ ID NO:317 D250R peptide Georgia 2007 / 1, 3-46, 10

[0499] SEQ ID NO:318 D250R protein BA71

[0500] SEQ ID NO:319 D250R protein Georgia 2007 / 1

[0501] SEQ ID NO:320 D339L peptide BA71

[0502] SEQ ID NO:321 D339L protein BA71

[0503] SEQ ID NO:322 D339L protein Georgia 2007 / 1

[0504] SEQ ID NO: 323 D345L peptide Georgia 2007 / 1, 125-133, 9

[0505] SEQ ID NO:324 D345L protein Georgia 2007 / 1

[0506] SEQ ID NO: 325 DP238L peptide BA71

[0507] SEQ ID NO:326 DP238L protein BA71

[0508] SEQ ID NO:327 DP238L protein Georgia 2007 / 1

[0509] SEQ ID NO: 328 DP96R peptide Georgia 2007 / 1, 46-55, 10

[0510] SEQ ID NO:329 DP96R protein Georgia 2007 / 1

[0511] SEQ ID NO:330 E111R peptide BA71

[0512] SEQ ID NO:331 E111R protein BA71

[0513] SEQ ID NO:332 E111R protein Georgia 2007 / 1

[0514] SEQ ID NO: 333 E120R peptide BA71

[0515] SEQ ID NO:334 E120R protein BA71

[0516] SEQ ID NO:335 E120R protein Georgia 2007 / 1

[0517] SEQ ID NO:336 E146L peptide Georgia 2007 / 1, 52-64, 13

[0518] SEQ ID NO:337 E146L peptide Georgia 2007 / 1, 55-64, 10

[0519] SEQ ID NO:338 E146L protein Georgia 2007 / 1

[0520] SEQ ID NO:339 E146L protein Georgia 2007 / 1

[0521] SEQ ID NO:340 E183L peptide BA71

[0522] SEQ ID NO:341 E183L protein BA71

[0523] SEQ ID NO:342 E183L protein Georgia 2007 / 1

[0524] SEQ ID NO: 343 E248R peptide BA71

[0525] SEQ ID NO:344 E248R protein BA71

[0526] SEQ ID NO:345 E248R protein Georgia 2007 / 1

[0527] SEQ ID NO:346 E301R peptide BA71

[0528] SEQ ID NO:347 E301R peptide Georgia 2007 / 1, 290-297 8

[0529] SEQ ID NO:348 E301R protein BA71

[0530] SEQ ID NO:349 E301R protein Georgia 2007 / 1

[0531] SEQ ID NO:350 E423R peptide Georgia 2007 / 1, 152-169, 18

[0532] SEQ ID NO:351 E423R peptide Georgia 2007 / 1, 57-64, 8

[0533] SEQ ID NO:352 E423R protein Georgia 2007 / 1

[0534] SEQ ID NO:353 E66L peptide Georgia 2007 / 1, 18-27, 10

[0535] SEQ ID NO:354 E66L peptide Georgia 2007 / 1, 4-13, 10

[0536] SEQ ID NO:355 E66L protein BA71

[0537] SEQ ID NO:356 E66L protein Georgia 2007 / 1

[0538] SEQ ID NO: 357 EP1242L peptide BA71

[0539] SEQ ID NO:358 EP1242L peptide BA71

[0540] SEQ ID NO: 359 EP1242L peptide Georgia 2007 / 1, 61-69, 9

[0541] SEQ ID NO: 360 EP1242L peptide Georgia 2007 / 1, 818-828, 11

[0542] SEQ ID NO:361 EP1242L protein BA71

[0543] SEQ ID NO:362 EP1242L protein Georgia 2007 / 1

[0544] SEQ ID NO: 363 EP152R peptide Georgia 2007 / 1, 40-60, 21

[0545] SEQ ID NO:364 EP152R protein Georgia 2007 / 1

[0546] SEQ ID NO: 365 EP153R peptide Georgia 2007 / 1, 116-124, 9

[0547] SEQ ID NO:366 EP153R protein Georgia 2007 / 1

[0548] SEQ ID NO: 367 EP296R peptide Georgia 2007 / 1, 235-245, 11

[0549] SEQ ID NO:368 EP296R protein Georgia 2007 / 1

[0550] SEQ ID NO: 369 EP364R peptide BA71

[0551] SEQ ID NO: 370 EP364R protein BA71

[0552] SEQ ID NO:371 EP364R protein Georgia 2007 / 1

[0553] SEQ ID NO: 372 EP402R peptide Georgia 2007 / 1, 150-158, 9

[0554] SEQ ID NO: 373 EP402R peptide Georgia 2007 / 1, 189-210, 22

[0555] SEQ ID NO: 374 EP402R peptide Georgia 2007 / 1, 190-200, 11

[0556] SEQ ID NO: 375 EP402R peptide Georgia 2007 / 1, 192-202, 11

[0557] SEQ ID NO: 376 EP402R peptide Georgia 2007 / 1, 196-206, 11

[0558] SEQ ID NO: 377 EP402R peptide Georgia 2007 / 1, 279-287, 9

[0559] SEQ ID NO:378 EP402R protein Georgia 2007 / 1

[0560] SEQ ID NO: 379 EP424R peptide BA71

[0561] SEQ ID NO: 380 EP424R peptide BA71

[0562] SEQ ID NO: 381 EP424R peptide Georgia 2007 / 1

[0563] SEQ ID NO:382 EP424R peptide Georgia 2007 / 1

[0564] SEQ ID NO: 383 EP424R peptide Georgia 2007 / 1, 137-146, 10

[0565] SEQ ID NO: 384 EP424R peptide Georgia 2007 / 1, 140-156, 17

[0566] SEQ ID NO: 385 EP424R peptide Georgia 2007 / 1, 146-154, 9

[0567] SEQ ID NO: 386 EP424R peptide Georgia 2007 / 1, 215-225, 11

[0568] SEQ ID NO: 387 EP424R peptide Georgia 2007 / 1, 270-278, 9

[0569] SEQ ID NO:388 EP424R protein BA71

[0570] SEQ ID NO:389 EP424R protein Georgia 2007 / 1

[0571] SEQ ID NO:390 F1055L peptide Georgia 2007 / 1, 111-127, 17

[0572] SEQ ID NO:391 F1055L peptide Georgia 2007 / 1, 112-121, 10

[0573] SEQ ID NO:392 F1055L peptide Georgia 2007 / 1, 116-125, 10

[0574] SEQ ID NO:393 F1055L peptide Georgia 2007 / 1, 527-535, 9

[0575] SEQ ID NO:394 F1055L peptide Georgia 2007 / 1, 905-923, 19

[0576] SEQ ID NO:395 F1055L protein Georgia 2007 / 1

[0577] SEQ ID NO:396 F165R peptide Georgia 2007 / 1, 14-36, 23

[0578] SEQ ID NO:397 F165R peptide Georgia 2007 / 1, 23-32, 10

[0579] SEQ ID NO:398 F165R protein Georgia 2007 / 1

[0580] SEQ ID NO:399 F317L peptide BA71

[0581] SEQ ID NO:400 F317L peptide BA71

[0582] SEQ ID NO:401 F317L peptide Georgia 2007 / 1, 246-256, 11

[0583] SEQ ID NO:402 F317L peptide Georgia 2007 / 1, 248-256, 9

[0584] SEQ ID NO:403 F317L peptide Georgia 2007 / 1, 4-13, 10

[0585] SEQ ID NO:404 F317L protein BA71

[0586] SEQ ID NO:405 F317L protein Georgia 2007 / 1

[0587] SEQ ID NO:406 F334L peptide BA71

[0588] SEQ ID NO:407 F334L peptide Georgia 2007 / 1, 264-274, 11

[0589] SEQ ID NO:408 F334L protein BA71

[0590] SEQ ID NO:409 F334L protein Georgia 2007 / 1

[0591] SEQ ID NO:410 F778R peptide Georgia 2007 / 1, 520-530, 11

[0592] SEQ ID NO: 411 F778R peptide Georgia 2007 / 1, 711-723, 13

[0593] SEQ ID NO:412 F778R peptide Georgia 2007 / 1, 712-720, 9

[0594] SEQ ID NO: 413 F778R peptide Georgia 2007 / 1, 713-723, 11

[0595] SEQ ID NO:414 F778R peptide Georgia 2007 / 1, 715-723, 9

[0596] SEQ ID NO:415 F778R protein Georgia 2007 / 1

[0597] SEQ ID NO:416 G1211R peptide BA71

[0598] SEQ ID NO:417 G1211R peptide BA71

[0599] SEQ ID NO:418 G1211R peptide BA71

[0600] SEQ ID NO:419 G1211R peptide BA71

[0601] SEQ ID NO:420 G1211R peptide Georgia 2007 / 1

[0602] SEQ ID NO:421 G1211R peptide Georgia 2007 / 1

[0603] SEQ ID NO:422 G1211R peptide Georgia 2007 / 1

[0604] SEQ ID NO:423 G1211R peptide Georgia 2007 / 1

[0605] SEQ ID NO:424 G1211R peptide Georgia 2007 / 1, 1043-1050, 8

[0606] SEQ ID NO:425 G1211R peptide Georgia 2007 / 1, 146-157, 12

[0607] SEQ ID NO:426 G1211R peptide Georgia 2007 / 1, 147-155, 9

[0608] SEQ ID NO:427 G1211R peptide Georgia 2007 / 1, 206-215, 10

[0609] SEQ ID NO:428 G1211R peptide Georgia 2007 / 1, 519-529, 11

[0610] SEQ ID NO:429 G1211R peptide Georgia 2007 / 1, 698-707, 10

[0611] SEQ ID NO:430 G1211R protein BA71

[0612] SEQ ID NO:431 G1211R protein BA71

[0613] SEQ ID NO:432 G1211R protein Georgia 2007 / 1

[0614] SEQ ID NO:433 G1211R protein Georgia 2007 / 1

[0615] SEQ ID NO:434 G1340L peptide BA71

[0616] SEQ ID NO:435 G1340L peptide Georgia 2007 / 1, 229-236, 8

[0617] SEQ ID NO:436 G1340L peptide Georgia 2007 / 1, 456-476, 21

[0618] SEQ ID NO:437 G1340L peptide Georgia 2007 / 1, 463-473, 11

[0619] SEQ ID NO:438 G1340L peptide Georgia 2007 / 1, 466-473, 8

[0620] SEQ ID NO:439 G1340L peptide Georgia 2007 / 1, 534-550, 17

[0621] SEQ ID NO:440 G1340L peptide Georgia 2007 / 1, 538-546, 9

[0622] SEQ ID NO:441 G1340L peptide Georgia 2007 / 1, 540-548, 9

[0623] SEQ ID NO:442 G1340L peptide Georgia 2007 / 1, 97-106, 10

[0624] SEQ ID NO:443 G1340L protein BA71

[0625] SEQ ID NO:444 G1340L protein Georgia 2007 / 1

[0626] SEQ ID NO:445 H124R peptide Georgia 2007 / 1, 51-61, 11

[0627] SEQ ID NO:446 H124R peptide Georgia 2007 / 1, 52-61, 10

[0628] SEQ ID NO:447 H124R protein Georgia 2007 / 1

[0629] SEQ ID NO:448 H233R peptide BA71

[0630] SEQ ID NO:449 H233R peptide BA71

[0631] SEQ ID NO:450 H233R protein BA71

[0632] SEQ ID NO:451 H233R protein Georgia 2007 / 1

[0633] SEQ ID NO:452 H240R peptide Georgia 2007 / 1, 139-148 10

[0634] SEQ ID NO:453 H240R protein Georgia 2007 / 1

[0635] SEQ ID NO:454 H339R peptide BA71

[0636] SEQ ID NO:455 H339R peptide BA71

[0637] SEQ ID NO:456 H339R peptide BA71

[0638] SEQ ID NO:457 H339R peptide BA71

[0639] SEQ ID NO:458 H339R peptide Georgia 2007 / 1

[0640] SEQ ID NO:459 H339R peptide Georgia 2007 / 1

[0641] SEQ ID NO:460 H339R peptide Georgia 2007 / 1

[0642] SEQ ID NO:461 H339R peptide Georgia 2007 / 1

[0643] SEQ ID NO:462 H339R peptide Georgia 2007 / 1

[0644] SEQ ID NO:463 H339R peptide Georgia 2007 / 1, 200-209, 10

[0645] SEQ ID NO: 464 H339R peptide Georgia 2007 / 1, 84-92, 9

[0646] SEQ ID NO:465 H339R peptide Georgia 2007 / 1, 87-95, 9

[0647] SEQ ID NO:466 H339R protein BA71

[0648] SEQ ID NO:467 H339R protein BA71

[0649] SEQ ID NO:468 H339R protein Georgia 2007 / 1

[0650] SEQ ID NO:469 H339R protein Georgia 2007 / 1

[0651] SEQ ID NO:470 H359L peptide BA71

[0652] SEQ ID NO:471 H359L protein BA71

[0653] SEQ ID NO:472 H359L protein Georgia 2007 / 1

[0654] SEQ ID NO:473 I10L peptide Georgia 2007 / 1, 136-146, 11

[0655] SEQ ID NO:474 I10L protein BA71

[0656] SEQ ID NO:475 I10L protein Georgia 2007 / 1

[0657] SEQ ID NO:476 I215L peptide Georgia 2007 / 1, 1-10, 10

[0658] SEQ ID NO:477 I215L protein Georgia 2007 / 1

[0659] SEQ ID NO:478 I226R peptide BA71

[0660] SEQ ID NO:479 I226R peptide BA71

[0661] SEQ ID NO:480 I226R peptide BA71

[0662] SEQ ID NO:481 I226R peptide Georgia 2007 / 1

[0663] SEQ ID NO:482 I226R peptide Georgia 2007 / 1

[0664] SEQ ID NO:483 I226R peptide Georgia 2007 / 1

[0665] SEQ ID NO:484 I226R peptide Georgia 2007 / 1

[0666] SEQ ID NO:485 I226R peptide Georgia 2007 / 1, 150-157, 8

[0667] SEQ ID NO:486 I226R peptide Georgia 2007 / 1, 51-58, 8

[0668] SEQ ID NO:487 I226R protein BA71

[0669] SEQ ID NO:488 I226R protein BA71

[0670] SEQ ID NO:489 I226R protein Georgia 2007 / 1

[0671] SEQ ID NO:490 I226R protein Georgia 2007 / 1

[0672] SEQ ID NO:491 I243L peptide BA71

[0673] SEQ ID NO:492 I243L peptide Georgia 2007 / 1, 81-89, 9

[0674] SEQ ID NO:493 I243L protein BA71

[0675] SEQ ID NO:494 I243L protein Georgia 2007 / 1

[0676] SEQ ID NO:495 I267L peptide Georgia 2007 / 1, 246-269, 24

[0677] SEQ ID NO:496 I267L peptide Georgia 2007 / 1, 252-260, 9

[0678] SEQ ID NO:497 I267L peptide Georgia 2007 / 1, 66-76, 11

[0679] SEQ ID NO:498 I267L protein Georgia 2007 / 1

[0680] SEQ ID NO:499 I329L peptide Georgia 2007 / 1, 136-145, 10

[0681] SEQ ID NO:500 I329L peptide Georgia 2007 / 1, 182-190, 9

[0682] SEQ ID NO:501 I329L protein Georgia 2007 / 1

[0683] SEQ ID NO:502 I73R peptide BA71

[0684] SEQ ID NO:503 I73R protein BA71

[0685] SEQ ID NO:504 I73R protein Georgia 2007 / 1

[0686] SEQ ID NO:505 I7L peptide Georgia 2007 / 1, 28-37, 10

[0687] SEQ ID NO:506 I7L protein Georgia 2007 / 1

[0688] SEQ ID NO:507 I8L peptide Georgia 2007 / 1, 88-95, 8

[0689] SEQ ID NO:508 I8L protein BA71

[0690] SEQ ID NO:509 I8L protein Georgia 2007 / 1

[0691] SEQ ID NO:510 I9R peptide BA7

[0692] SEQ ID NO:511 I9R peptide Georgia 2007 / 1, 63-73, 11

[0693] SEQ ID NO:512 I9R protein BA71

[0694] SEQ ID NO:513 I9R protein Georgia 2007 / 1

[0695] SEQ ID NO:514 K145R peptide BA71

[0696] SEQ ID NO:515 K145R peptide BA71

[0697] SEQ ID NO:516 K145R peptide BA71

[0698] SEQ ID NO:517 K145R peptide BA71

[0699] SEQ ID NO:518 K145R peptide Georgia 2007 / 1

[0700] SEQ ID NO:519 K145R peptide Georgia 2007 / 1

[0701] SEQ ID NO:520 K145R peptide Georgia 2007 / 1

[0702] SEQ ID NO:521 K145R peptide Georgia 2007 / 1

[0703] SEQ ID NO:522 K145R peptide Georgia 2007 / 1, 43-58, 16

[0704] SEQ ID NO:523 K145R peptide Georgia 2007 / 1, 48-57, 10

[0705] SEQ ID NO:524 K145R protein BA71

[0706] SEQ ID NO:525 K145R protein BA71

[0707] SEQ ID NO:526 K145R protein Georgia 2007 / 1

[0708] SEQ ID NO:527 K145R protein Georgia 2007 / 1

[0709] SEQ ID NO:528 K205R peptide Georgia 2007 / 1, 38-48, 11

[0710] SEQ ID NO:529 K205R protein Georgia 2007 / 1

[0711] SEQ ID NO:530 K421R peptide Georgia 2007 / 1, 69-86, 18

[0712] SEQ ID NO:531 K421R peptide Georgia 2007 / 1, 78-86, 9

[0713] SEQ ID NO:532 K421R protein Georgia 2007 / 1

[0714] SEQ ID NO:533 K78R peptide Georgia 2007 / 1, 36-44, 9

[0715] SEQ ID NO:534 K78R protein Georgia 2007 / 1

[0716] SEQ ID NO:535 L11L protein BA71

[0717] SEQ ID NO:536 L11L protein Georgia 2007 / 1

[0718] SEQ ID NO:537 L60L protein BA71

[0719] SEQ ID NO:538 L60L protein Georgia 2007 / 1

[0720] SEQ ID NO:539 M1249L peptide BA71

[0721] SEQ ID NO:540 M1249L peptide BA71

[0722] SEQ ID NO:541 M1249L peptide BA71

[0723] SEQ ID NO:542 M1249L peptide Georgia 2007 / 1

[0724] SEQ ID NO:543 M1249L peptide Georgia 2007 / 1

[0725] SEQ ID NO:544 M1249L peptide Georgia 2007 / 1

[0726] SEQ ID NO:545 M1249L peptide Georgia 2007 / 1, 1068-1078, 11

[0727] SEQ ID NO:546 M1249L peptide Georgia 2007 / 1, 1130-1140, 11

[0728] SEQ ID NO:547 M1249L peptide Georgia 2007 / 1, 1130-1144, 15

[0729] SEQ ID NO:548 M1249L peptide Georgia 2007 / 1, 266-275, 10

[0730] SEQ ID NO:549 M1249L peptide Georgia 2007 / 1, 266-292, 27

[0731] SEQ ID NO:550 M1249L peptide Georgia 2007 / 1, 270-279, 10

[0732] SEQ ID NO:551 M1249L peptide Georgia 2007 / 1, 276-284, 9

[0733] SEQ ID NO:552 M1249L peptide Georgia 2007 / 1, 282-291, 10

[0734] SEQ ID NO:553 M1249L peptide Georgia 2007 / 1, 555-564, 10

[0735] SEQ ID NO:554 M1249L peptide Georgia 2007 / 1, 583-590, 8

[0736] SEQ ID NO:555 M1249L peptide Georgia 2007 / 1, 772-782, 11

[0737] SEQ ID NO:556 M1249L peptide Georgia 2007 / 1, 778-789, 12

[0738] SEQ ID NO:557 M1249L peptide Georgia 2007 / 1, 780-788, 9

[0739] SEQ ID NO:558 M1249L peptide Georgia 2007 / 1, 917-933, 17

[0740] SEQ ID NO:559 M1249L peptide Georgia 2007 / 1, 922-932, 11

[0741] SEQ ID NO:560 M1249L peptide Georgia 2007 / 1, 923-933, 11

[0742] SEQ ID NO:561 M1249L protein BA71

[0743] SEQ ID NO:562 M1249L protein Georgia 2007 / 1

[0744] SEQ ID NO:563 M448R peptide BA71

[0745] SEQ ID NO:564 M448R peptide Georgia 2007 / 1, 146-156, 11

[0746] SEQ ID NO:565 M448R peptide Georgia 2007 / 1, 248-257, 10

[0747] SEQ ID NO:566 M448R protein BA71

[0748] SEQ ID NO:567 M448R protein BA71

[0749] SEQ ID NO:568 M448R protein Georgia 2007 / 1

[0750] SEQ ID NO:569 M448R protein Georgia 2007 / 1

[0751] SEQ ID NO:570 MGF_100-1L peptide Georgia 2007 / 1, 68-86, 19

[0752] SEQ ID NO:571 MGF_100-1L peptide Georgia 2007 / 1, 68-86, 19

[0753] SEQ ID NO:572 MGF_100-1L protein Georgia 2007 / 1

[0754] SEQ ID NO:573 MGF_100-1L protein Georgia 2007 / 1

[0755] SEQ ID NO:574 MGF_100-1R peptide Georgia 2007 / 1, 103-113, 11

[0756] SEQ ID NO:575 MGF_100-1R peptide Georgia 2007 / 1, 97-115, 19

[0757] SEQ ID NO:576 MGF_100-1R protein Georgia 2007 / 1

[0758] SEQ ID NO:577 MGF_110-11L peptide Georgia 2007 / 1, 84-94, 11

[0759] SEQ ID NO:578 MGF_110-11L protein Georgia 2007 / 1

[0760] SEQ ID NO:579 MGF_110-12L peptide Georgia 2007 / 1, 19-29, 11

[0761] SEQ ID NO:580 MGF_110-12L protein Georgia 2007 / 1

[0762] SEQ ID NO:581 MGF_110-14L peptide Georgia 2007 / 1, 72-82, 11

[0763] SEQ ID NO:582 MGF_110-14L protein Georgia 2007 / 1

[0764] SEQ ID NO:583 MGF_110-1L peptide Georgia 2007 / 1, 10-19, 10

[0765] SEQ ID NO:584 MGF_110-1L peptide Georgia 2007 / 1, 77-87, 11

[0766] SEQ ID NO:585 MGF_110-1L protein Georgia 2007 / 1

[0767] SEQ ID NO:586 MGF_110-6L peptide BA71

[0768] SEQ ID NO:587 MGF_110-6L protein BA71

[0769] SEQ ID NO:588 MGF_110-6L protein Georgia 2007 / 1

[0770] SEQ ID NO:589 MGF_110-8L peptide Georgia 2007 / 1, 81-91, 11

[0771] SEQ ID NO:590 MGF_110-8L protein Georgia 2007 / 1

[0772] SEQ ID NO:591 MGF_110-9L peptide Georgia 2007 / 1, 101-110, 10

[0773] SEQ ID NO:592 MGF_110-9L peptide Georgia 2007 / 1, 101-118, 18

[0774] SEQ ID NO:593 MGF_110-9L peptide Georgia 2007 / 1, 138-147, 10

[0775] SEQ ID NO:594 MGF_110-9L peptide Georgia 2007 / 1, 140-147, 8

[0776] SEQ ID NO:595 MGF_110-9L peptide Georgia 2007 / 1, 150-167, 18

[0777] SEQ ID NO:596 MGF_110-9L peptide Georgia 2007 / 1, 157-167, 11

[0778] SEQ ID NO:597 MGF_110-9L peptide Georgia 2007 / 1, 252-262, 11

[0779] SEQ ID NO:598 MGF_110-9L protein Georgia 2007 / 1

[0780] SEQ ID NO:599 MGF_300-1L peptide Georgia 2007 / 1, 172-190, 19

[0781] SEQ ID NO:600 MGF_300-1L peptide Georgia 2007 / 1, 179-189, 11

[0782] SEQ ID NO:601 MGF_300-1L peptide Georgia 2007 / 1, 78-87, 10

[0783] SEQ ID NO:602 MGF_300-1L protein Georgia 2007 / 1

[0784] SEQ ID NO:603 MGF_300-4L peptide Georgia 2007 / 1, 163-174, 12

[0785] SEQ ID NO:604 MGF_300-4L peptide Georgia 2007 / 1, 165-172, 8

[0786] SEQ ID NO:605 MGF_300-4L protein Georgia 2007 / 1

[0787] SEQ ID NO:606 MGF_360-10L peptide BA71

[0788] SEQ ID NO:607 MGF_360-10L peptide Georgia 2007 / 1, 136-146, 11

[0789] SEQ ID NO:608 MGF_360-10L peptide Georgia 2007 / 1, 195-206, 12

[0790] SEQ ID NO:609 MGF_360-10L peptide Georgia 2007 / 1, 266-274, 9

[0791] SEQ ID NO:610 MGF_360-10L protein BA71

[0792] SEQ ID NO:611 MGF_360-10L protein Georgia 2007 / 1

[0793] SEQ ID NO:612 MGF_360-11L peptide Georgia 2007 / 1, 221-229, 9

[0794] SEQ ID NO:613 MGF_360-11L peptide Georgia 2007 / 1, 240-260, 21

[0795] SEQ ID NO:614 MGF_360-11L peptide Georgia 2007 / 1, 247-257, 11

[0796] SEQ ID NO:615 MGF_360-11L protein Georgia 2007 / 1

[0797] SEQ ID NO:616 MGF_360-12L peptide Georgia 2007 / 1, 165-186, 22

[0798] SEQ ID NO:617 MGF_360-12L peptide Georgia 2007 / 1, 174-182, 9

[0799] SEQ ID NO:618 MGF_360-12L peptide Georgia 2007 / 1, 174-184, 11

[0800] SEQ ID NO:619 MGF_360-12L peptide Georgia 2007 / 1, 266-274, 9

[0801] SEQ ID NO:620 MGF_360-12L protein Georgia 2007 / 1

[0802] SEQ ID NO:621 MGF_360-13L peptide Georgia 2007 / 1, 271-281, 11

[0803] SEQ ID NO:622 MGF_360-13L protein Georgia 2007 / 1

[0804] SEQ ID NO:623 MGF_360-14L peptide Georgia 2007 / 1, 195-206, 12

[0805] SEQ ID NO:624 MGF_360-14L protein Georgia 2007 / 1

[0806] SEQ ID NO:625 MGF_360-15R peptide Georgia 2007 / 1, 16-25, 10

[0807] SEQ ID NO:626 MGF_360-15R peptide Georgia 2007 / 1, 37-46, 10

[0808] SEQ ID NO:627 MGF_360-15R peptide Georgia 2007 / 1, 71-81, 11

[0809] SEQ ID NO:628 MGF_360-15R protein Georgia 2007 / 1

[0810] SEQ ID NO:629 MGF_360-18R peptide Georgia 2007 / 1, 159-167, 9

[0811] SEQ ID NO:630 MGF_360-18R peptide Georgia 2007 / 1, 93-100, 8

[0812] SEQ ID NO:631 MGF_360-18R protein Georgia 2007 / 1

[0813] SEQ ID NO:632 MGF_360-1L peptide Georgia 2007 / 1, 184-192, 9

[0814] SEQ ID NO:633 MGF_360-1L peptide Georgia 2007 / 1, 188-198, 11

[0815] SEQ ID NO:634 MGF_360-1L peptide Georgia 2007 / 1, 192-200, 9

[0816] SEQ ID NO:635 MGF_360-1L peptide Georgia 2007 / 1, 195-203, 9

[0817] SEQ ID NO:636 MGF_360-1L peptide Georgia 2007 / 1, 244-254, 11

[0818] SEQ ID NO:637 MGF_360-1L protein Georgia 2007 / 1

[0819] SEQ ID NO:638 MGF_360-21R peptide Georgia 2007 / 1, 176-185, 10

[0820] SEQ ID NO:639 MGF_360-21R peptide Georgia 2007 / 1, 177-187, 11

[0821] SEQ ID NO:640 MGF_360-21R peptide Georgia 2007 / 1, 185-193, 9

[0822] SEQ ID NO:641 MGF_360-21R peptide Georgia 2007 / 1, 189-199, 11

[0823] SEQ ID NO:642 MGF_360-21R peptide Georgia 2007 / 1, 197-207, 11

[0824] SEQ ID NO:643 MGF_360-21R peptide Georgia 2007 / 1, 246-256, 11

[0825] SEQ ID NO:644 MGF_360-21R protein Georgia 2007 / 1

[0826] SEQ ID NO:645 MGF_360-2L peptide Georgia 2007 / 1, 191-198, 8

[0827] SEQ ID NO:646 MGF_360-2L peptide Georgia 2007 / 1, 191-207, 17

[0828] SEQ ID NO:647 MGF_360-2L peptide Georgia 2007 / 1, 196-203, 8

[0829] SEQ ID NO:648 MGF_360-2L protein Georgia 2007 / 1

[0830] SEQ ID NO:649 MGF_360-3L peptide Georgia 2007 / 1, 167-186, 20

[0831] SEQ ID NO:650 MGF_360-3L protein Georgia 2007 / 1

[0832] SEQ ID NO:651 MGF_360-4L peptide Georgia 2007 / 1, 162-170, 9

[0833] SEQ ID NO:652 MGF_360-4L peptide Georgia 2007 / 1, 363-372, 10

[0834] SEQ ID NO:653 MGF_360-4L protein Georgia 2007 / 1

[0835] SEQ ID NO:654 MGF_360-6L peptide Georgia 2007 / 1, 150-162, 13

[0836] SEQ ID NO: 655 MGF_360-6L peptide Georgia 2007 / 1, 356-364, 9

[0837] SEQ ID NO:656 MGF_360-6L protein Georgia 2007 / 1

[0838] SEQ ID NO:657 MGF_360-8L peptide BA71

[0839] SEQ ID NO:658 MGF_360-8L peptide BA71

[0840] SEQ ID NO:659 MGF_360-8L peptide Georgia 2007 / 1, 189-198, 10

[0841] SEQ ID NO:660 MGF_360-8L peptide Georgia 2007 / 1, 195-206, 12

[0842] SEQ ID NO:661 MGF_360-8L peptide Georgia 2007 / 1, 196-206, 11

[0843] SEQ ID NO:662 MGF_360-8L peptide Georgia 2007 / 1, 80-90, 11

[0844] SEQ ID NO:663 MGF_360-8L protein BA71

[0845] SEQ ID NO:664 MGF_360-8L protein Georgia 2007 / 1

[0846] SEQ ID NO:665 MGF_360-9L peptide Georgia 2007 / 1, 193-203, 11

[0847] SEQ ID NO:666 MGF_360-9L peptide Georgia 2007 / 1, 257-267, 11

[0848] SEQ ID NO:667 MGF_360-9L peptide Georgia 2007 / 1, 257-269, 13

[0849] SEQ ID NO:668 MGF_360-9L peptide Georgia 2007 / 1, 259-267, 9

[0850] SEQ ID NO:669 MGF_360-9L peptide Georgia 2007 / 1, 264-274, 11

[0851] SEQ ID NO:670 MGF_360-9L peptide Georgia 2007 / 1, 300-307, 8

[0852] SEQ ID NO:671 MGF_360-9L protein Georgia 2007 / 1

[0853] SEQ ID NO:672 MGF_505-10R peptide Georgia 2007 / 1,499-509,11

[0854] SEQ ID NO:673 MGF_505-10R protein Georgia 2007 / 1

[0855] SEQ ID NO:674 MGF_505-11L peptide Georgia 2007 / 1, 111-129, 19

[0856] SEQ ID NO:675 MGF_505-11L peptide Georgia 2007 / 1, 142-149, 8

[0857] SEQ ID NO:676 MGF_505-11L peptide Georgia 2007 / 1, 230-247, 18

[0858] SEQ ID NO:677 MGF_505-11L peptide Georgia 2007 / 1, 231-241, 11

[0859] SEQ ID NO:678 MGF_505-11L peptide Georgia 2007 / 1, 308-327, 20

[0860] SEQ ID NO:679 MGF_505-11L peptide Georgia 2007 / 1, 312-322, 11

[0861] SEQ ID NO:680 MGF_505-11L peptide Georgia 2007 / 1, 315-322, 8

[0862] SEQ ID NO:681 MGF_505-11L peptide Georgia 2007 / 1, 319-327, 9

[0863] SEQ ID NO:682 MGF_505-11L peptide Georgia 2007 / 1, 528-538, 11

[0864] SEQ ID NO:683 MGF_505-11L protein Georgia 2007 / 1

[0865] SEQ ID NO:684 MGF_505-1R peptide BA71

[0866] SEQ ID NO:685 MGF_505-1R peptide BA71

[0867] SEQ ID NO:686 MGF_505-1R peptide Georgia 2007 / 1

[0868] SEQ ID NO:687 MGF_505-1R peptide Georgia 2007 / 1

[0869] SEQ ID NO:688 MGF_505-1R peptide Georgia 2007 / 1, 270-280, 11

[0870] SEQ ID NO:689 MGF_505-1R peptide Georgia 2007 / 1, 427-437, 11

[0871] SEQ ID NO:690 MGF_505-1R peptide Georgia 2007 / 1, 76-85, 10

[0872] SEQ ID NO:691 MGF_505-1R protein BA71

[0873] SEQ ID NO:692 MGF_505-1R protein Georgia 2007 / 1

[0874] SEQ ID NO:693 MGF_505-2R peptide BA71

[0875] SEQ ID NO:694 MGF_505-2R peptide Georgia 2007 / 1, 160-168, 9

[0876] SEQ ID NO:695 MGF_505-2R peptide Georgia 2007 / 1, 197-206, 10

[0877] SEQ ID NO:696 MGF_505-2R peptide Georgia 2007 / 1, 311-319, 9

[0878] SEQ ID NO:697 MGF_505-2R protein BA71

[0879] SEQ ID NO:698 MGF_505-2R protein Georgia 2007 / 1

[0880] SEQ ID NO:699 MGF_505-3R peptide BA71

[0881] SEQ ID NO:700 MGF_505-3R peptide Georgia 2007 / 1, 75-85, 11

[0882] SEQ ID NO:701 MGF_505-3R peptide Georgia 2007 / 1, 86-95, 10

[0883] SEQ ID NO:702 MGF_505-3R protein BA71

[0884] SEQ ID NO:703 MGF_505-3R protein Georgia 2007 / 1

[0885] SEQ ID NO:704 MGF_505-4R peptide Georgia 2007 / 1, 204-212, 9

[0886] SEQ ID NO:705 MGF_505-4R peptide Georgia 2007 / 1, 352-362, 11

[0887] SEQ ID NO:706 MGF_505-4R protein Georgia 2007 / 1

[0888] SEQ ID NO:707 MGF_505-5R peptide BA71

[0889] SEQ ID NO:708 MGF_505-5R peptide Georgia 2007 / 1, 200-208, 9

[0890] SEQ ID NO:709 MGF_505-5R peptide Georgia 2007 / 1, 204-212, 9

[0891] SEQ ID NO:710 MGF_505-5R peptide Georgia 2007 / 1, 346-356, 11

[0892] SEQ ID NO:711 MGF_505-5R peptide Georgia 2007 / 1, 354-366, 13

[0893] SEQ ID NO:712 MGF_505-5R protein BA71

[0894] SEQ ID NO:713 MGF_505-5R protein Georgia 2007 / 1

[0895] SEQ ID NO:714 MGF_505-6R peptide Georgia 2007 / 1, 102-111, 10

[0896] SEQ ID NO:715 MGF_505-6R peptide Georgia 2007 / 1, 427-435, 9

[0897] SEQ ID NO:716 MGF_505-6R protein Georgia 2007 / 1

[0898] SEQ ID NO:717 MGF_505-8R peptide BA71

[0899] SEQ ID NO:718 MGF_505-8R peptide BA71

[0900] SEQ ID NO:719 MGF_505-7R peptide Georgia 2007 / 1

[0901] SEQ ID NO:720 MGF_505-7R peptide Georgia 2007 / 1

[0902] SEQ ID NO:721 MGF_505-7R peptide Georgia 2007 / 1, 102-111, 10

[0903] SEQ ID NO:722 MGF_505-8R protein BA71 (A469R)

[0904] SEQ ID NO:723 MGF_505-8R protein BA71 (A469R)

[0905] SEQ ID NO:724 MGF_505-7R protein Georgia 2007 / 1

[0906] SEQ ID NO:725 MGF_505-7R protein Georgia 2007 / 1

[0907] SEQ ID NO:726 MGF_505-9R peptide BA71

[0908] SEQ ID NO:727 MGF_505-9R peptide Georgia 2007 / 1

[0909] SEQ ID NO:728 MGF_505-9R peptide Georgia 2007 / 1

[0910] SEQ ID NO:729 MGF_505-9R peptide Georgia 2007 / 1

[0911] SEQ ID NO:730 MGF_505-9R peptide Georgia 2007 / 1

[0912] SEQ ID NO:731 MGF_505-9R peptide Georgia 2007 / 1, 355-363, 9

[0913] SEQ ID NO:732 MGF_505-9R protein BA71

[0914] SEQ ID NO:733 MGF_505-9R protein Georgia 2007 / 1

[0915] SEQ ID NO:734 MGF110-12L protein BA71

[0916] SEQ ID NO:735 MGF110-12L protein Georgia 2007 / 1

[0917] SEQ ID NO:736 MGF110-13L protein BA71

[0918] SEQ ID NO:737 MGF110-13L protein Georgia 2007 / 1

[0919] SEQ ID NO:738 MGF110-14L protein BA71

[0920] SEQ ID NO:739 MGF110-14L protein Georgia 2007 / 1

[0921] SEQ ID NO:740 MGF110-1L protein BA71

[0922] SEQ ID NO:741 MGF110-1L protein Georgia2007 / 1

[0923] SEQ ID NO:742 MGF300-1L protein BA71

[0924] SEQ ID NO:743 MGF300-1L protein Georgia2007 / 1

[0925] SEQ ID NO:744 MGF300-4L protein BA71

[0926] SEQ ID NO:745 MGF300-4L protein Georgia 2007 / 1

[0927] SEQ ID NO:746 MGF360-10L protein BA71

[0928] SEQ ID NO:747 MGF360-10L protein Georgia 2007 / 1

[0929] SEQ ID NO:748 MGF360-11L protein BA71

[0930] SEQ ID NO:749 MGF360-11L protein Georgia 2007 / 1

[0931] SEQ ID NO:750 MGF360-12L protein BA71

[0932] SEQ ID NO:751 MGF360-12L protein Georgia 2007 / 1

[0933] SEQ ID NO:752 MGF360-14L protein BA71

[0934] SEQ ID NO:753 MGF360-14L protein Georgia 2007 / 1

[0935] SEQ ID NO:754 MGF360-15R protein BA71

[0936] SEQ ID NO:755 MGF360-15R protein Georgia 2007 / 1

[0937] SEQ ID NO:756 MGF360-2L protein BA71

[0938] SEQ ID NO:757 MGF360-2L protein Georgia 2007 / 1

[0939] SEQ ID NO:758 MGF360-3L protein BA71

[0940] SEQ ID NO:759 MGF360-3L protein Georgia 2007 / 1

[0941] SEQ ID NO:760 MGF360-4L protein BA71

[0942] SEQ ID NO:761 MGF360-4L protein Georgia 2007 / 1

[0943] SEQ ID NO:762 MGF360-6L protein BA71

[0944] SEQ ID NO:763 MGF360-6L protein Georgia 2007 / 1

[0945] SEQ ID NO:764 MGF360-8L protein BA71

[0946] SEQ ID NO:765 MGF360-8L protein Georgia 2007 / 1

[0947] SEQ ID NO:766 MGF505-2R protein BA71

[0948] SEQ ID NO:767 MGF505-2R protein Georgia 2007 / 1

[0949] SEQ ID NO:768 MGF505-4R protein BA71

[0950] SEQ ID NO:769 MGF505-4R protein Georgia 2007 / 1

[0951] SEQ ID NO:770 MGF505-7R protein BA71 (A528R)

[0952] SEQ ID NO:771 MGF505-6R protein Georgia 2007 / 1

[0953] SEQ ID NO:772 MGF505-8R protein BA71

[0954] SEQ ID NO:773 MGF505-8R protein BA71

[0955] SEQ ID NO:774 MGF505-7R protein Georgia 2007 / 1

[0956] SEQ ID NO:775 MGF505-7R protein Georgia 2007 / 1

[0957] SEQ ID NO:776 NP1450L peptide BA71

[0958] SEQ ID NO: 777 NP1450L peptide Georgia 2007 / 1, 1008-1016, 9

[0959] SEQ ID NO:778 NP1450L peptide Georgia 2007 / 1, 102-110, 9

[0960] SEQ ID NO:779 NP1450L peptide Georgia 2007 / 1, 1032-1040, 9

[0961] SEQ ID NO:780 NP1450L peptide Georgia 2007 / 1, 1136-1146, 11

[0962] SEQ ID NO:781 NP1450L peptide Georgia 2007 / 1, 1327-1336, 10

[0963] SEQ ID NO:782 NP1450L peptide Georgia 2007 / 1, 575-587, 13

[0964] SEQ ID NO:783 NP1450L peptide Georgia 2007 / 1, 645-653, 9

[0965] SEQ ID NO:784 NP1450L peptide Georgia 2007 / 1, 776-784, 9

[0966] SEQ ID NO:785 NP1450L protein BA71

[0967] SEQ ID NO:786 NP1450L protein Georgia 2007 / 1

[0968] SEQ ID NO:787 NP419L peptide BA71

[0969] SEQ ID NO:788 NP419L peptide Georgia 2007 / 1, 355-363, 9

[0970] SEQ ID NO:789 NP419L peptide Georgia 2007 / 1, 363-378, 16

[0971] SEQ ID NO:790 NP419L protein BA71

[0972] SEQ ID NO:791 NP419L protein Georgia 2007 / 1

[0973] SEQ ID NO:792 NP868R peptide Georgia 2007 / 1, 176-186, 11

[0974] SEQ ID NO:793 NP868R peptide Georgia 2007 / 1, 184-192, 9

[0975] SEQ ID NO:794 NP868R peptide Georgia 2007 / 1, 495-515, 21

[0976] SEQ ID NO:795 NP868R peptide Georgia 2007 / 1, 570-579, 10

[0977] SEQ ID NO:796 NP868R protein Georgia 2007 / 1

[0978] SEQ ID NO:797 O174L peptide Georgia 2007 / 1,97-105,9

[0979] SEQ ID NO:798 O174L protein Georgia 2007 / 1

[0980] SEQ ID NO:799 O61R peptide Georgia 2007 / 1, 23-38, 16

[0981] SEQ ID NO:800 O61R protein Georgia 2007 / 1

[0982] SEQ ID NO:801 P1192R peptide BA71

[0983] SEQ ID NO:802 P1192R peptide BA71

[0984] SEQ ID NO:803 P1192R peptide BA71

[0985] SEQ ID NO:804 P1192R peptide Georgia 2007 / 1

[0986] SEQ ID NO:805 P1192R peptide Georgia 2007 / 1

[0987] SEQ ID NO:806 P1192R peptide Georgia 2007 / 1, 1023-1040, 18

[0988] SEQ ID NO:807 P1192R peptide Georgia 2007 / 1, 1026-1033, 8

[0989] SEQ ID NO:808 P1192R peptide Georgia 2007 / 1, 1031-1038, 8

[0990] SEQ ID NO:809 P1192R peptide Georgia 2007 / 1, 1049-1056, 8

[0991] SEQ ID NO:810 P1192R peptide Georgia 2007 / 1, 1057-1067, 11

[0992] SEQ ID NO:811 P1192R peptide Georgia 2007 / 1, 189-197, 9

[0993] SEQ ID NO:812 P1192R peptide Georgia 2007 / 1, 372-382, 11

[0994] SEQ ID NO:813 P1192R peptide Georgia 2007 / 1, 584-592, 9

[0995] SEQ ID NO:814 P1192R peptide Georgia 2007 / 1, 605-614, 10

[0996] SEQ ID NO:815 P1192R peptide Georgia 2007 / 1, 744-751, 8

[0997] SEQ ID NO:816 P1192R protein BA71

[0998] SEQ ID NO:817 P1192R protein Georgia 2007 / 1

[0999] SEQ ID NO:818 Q706L peptide BA71

[1000] SEQ ID NO:819 Q706L peptide Georgia 2007 / 1, 137-144, 8

[1001] SEQ ID NO:820 Q706L peptide Georgia 2007 / 1,36-46,11

[1002] SEQ ID NO:821 Q706L peptide Georgia 2007 / 1,47-55,9

[1003] SEQ ID NO:822 Q706L protein BA71

[1004] SEQ ID NO:823 Q706L protein Georgia 2007 / 1

[1005] SEQ ID NO:824 QP383R peptide BA71

[1006] SEQ ID NO:825 QP383R peptide BA71

[1007] SEQ ID NO:826 QP383R peptide BA71

[1008] SEQ ID NO:827 QP383R protein BA71

[1009] SEQ ID NO:828 QP383R protein Georgia 2007 / 1

[1010] SEQ ID NO:829 QP509L peptide Georgia 2007 / 1, 384-402, 19

[1011] SEQ ID NO:830 QP509L peptide Georgia 2007 / 1, 392-400, 9

[1012] SEQ ID NO:831 QP509L peptide Georgia 2007 / 1, 51-63, 13

[1013] SEQ ID NO:832 QP509L peptide Georgia 2007 / 1, 76-85, 10

[1014] SEQ ID NO:833 QP509L protein Georgia 2007 / 1

[1015] SEQ ID NO:834 R298L 1 protein Georgia 2007 / 1

[1016] SEQ ID NO:835 R298L peptide Georgia 2007 / 1, 189-199, 11

[1017] SEQ ID NO:836 R298L peptide Georgia 2007 / 1, 189-214, 26

[1018] SEQ ID NO:837 R298L peptide Georgia 2007 / 1, 193-201, 9

[1019] SEQ ID NO:838 R298L peptide Georgia 2007 / 1, 193-203, 11

[1020] SEQ ID NO:839 R298L peptide Georgia 2007 / 1, 198-207, 10

[1021] SEQ ID NO:840 R298L peptide Georgia 2007 / 1, 205-214, 10

[1022] SEQ ID NO:841 R298L peptide Georgia 2007 / 1, 220-230, 11

[1023] SEQ ID NO:842 R298L peptide Georgia 2007 / 1, 99-108, 10

[1024] SEQ ID NO:843 R298L protein Georgia 2007 / 1

[1025] SEQ ID NO:844 S183L peptide Georgia 2007 / 1, 10-18, 9

[1026] SEQ ID NO:845 S183L peptide Georgia 2007 / 1, 128-136, 9

[1027] SEQ ID NO:846 S183L protein Georgia 2007 / 1

[1028] SEQ ID NO:847 S273R peptide Georgia 2007 / 1, 113-122, 10

[1029] SEQ ID NO:848 S273R peptide Georgia 2007 / 1, 96-104, 9

[1030] SEQ ID NO:849 S273R protein Georgia 2007 / 1

[1031] SEQ ID NO:850 X69R peptide Georgia 2007 / 1, 19-29, 11

[1032] SEQ ID NO:851 X69R peptide Georgia 2007 / 1, 47-54, 8

[1033] SEQ ID NO:852 X69R protein Georgia 2007 / 1

[1034] SEQ ID NO:853 A240L protein BA71

[1035] SEQ ID NO:854 A240L protein Georgia 2007 / 1

[1036] SEQ ID NO:855 Multi-epitope-1 (ME-1) construct

[1037] SEQ ID NO:856 Multi-epitope-II (ME-II) construct

[1038] SEQ ID NO:857 MGF505-7R protein Georgia 2007 / 1

[1039] SEQ ID NO:858 M448R protein Georgia 2007 / 1

[1040] SEQ ID NO:859 D1133L protein Georgia 2007 / 1

[1041] SEQ ID NO:860 A240L protein Georgia 2007 / 1

[1042] SEQ ID NO:861 CP312R protein Georgia 2007 / 1

[1043] SEQ ID NO:862 D117L protein Georgia 2007 / 1

[1044] SEQ ID NO:863 D117L protein Georgia 2007 / 1

[1045] SEQ ID NO:864 D117L protein BA71

[1046] SEQ ID NO:865 D117L protein BA71

[1047] SEQ ID NO:866 B475L protein Georgia 2007 / 1

[1048] SEQ ID NO:867 B475L protein BA71

[1049] SEQ ID NO:868 CP2475L (p37) protein Georgia 2007 / 1

[1050] SEQ ID NO:869 CP2475L (p37) protein BA71

[1051] SEQ ID NO:870 CP2475L (p150) protein Georgia 2007 / 1

[1052] SEQ ID NO:871 CP2475L (p150) protein BA71

[1053] SEQ ID NO:872 M1249L protein Georgia 2007 / 1

[1054] SEQ ID NO:873 M1249L protein BA71

[1055] SEQ ID NO:874 MGF-505-9R protein Georgia 2007 / 1

[1056] SEQ ID NO:875 MGF-505-9R protein BA71

[1057] SEQ ID NO:876 P1192R protein Georgia 2007 / 1

[1058] SEQ ID NO:877 P1192R protein BA71

[1059] SEQ ID NO:878 MGF-505-1R protein Georgia 2007 / 1

[1060] SEQ ID NO:879 MGF-505-1R protein BA71

[1061] SEQ ID NO:880 MGF-505-3R protein Georgia 2007 / 1

[1062] SEQ ID NO:881 MGF-505-3R protein BA71

[1063] SEQ ID NO:882 EP424R protein Georgia 2007 / 1

[1064] SEQ ID NO:883 EP424R protein BA71

[1065] SEQ ID NO:884 C475L protein Georgia 2007 / 1

[1066] SEQ ID NO:885 C475L protein BA71

[1067] SEQ ID NO:886 B602L protein Georgia 2007 / 1

[1068] SEQ ID NO:887 B602L protein BA71

[1069] SEQ ID NO:888 CP530R protein Georgia 2007 / 1

[1070] SEQ ID NO:889 CP530R protein BA71

[1071] SEQ ID NO:890 D339L protein Georgia 2007 / 1

[1072] SEQ ID NO:891 D339L protein BA71

[1073] SEQ ID NO:892 I234L protein Georgia 2007 / 1

[1074] SEQ ID NO:893 I234L protein BA71

[1075] SEQ ID NO:894 I73R protein Georgia 2007 / 1

[1076] SEQ ID NO:895 I73R protein BA71

[1077] SEQ ID NO:896 DP238L protein Georgia 2007 / 1

[1078] SEQ ID NO:897 DP238L protein BA71

[1079] SEQ ID NO:898 I9R protein Georgia 2007 / 1

[1080] SEQ ID NO:899 I9R protein BA71

[1081] SEQ ID NO:900 CP312R peptide Georgia 2007 / 1

[1082] SEQ ID NO:901 CP312R peptide Georgia 2007 / 1

[1083] Example

[1084] Example 1 - Materials and Methods

[1085] Cells and viruses

[1086] Porcine alveolar macrophages: Porcine alveolar macrophages (PAMs) from healthy common pigs (Landrace x Large White) were obtained by lung lavage with PBS 1× supplemented with 1 μg / ml gentamicin (Sigma-Aldrich). PBS solution was administered through the trachea using a sterile funnel, the lung lobes were gently massaged for 5 minutes, and the volume was collected into a sterile container. After washing three times with 250 ml of PBS solution, the recovered fluid was centrifuged at 400×g for 10 minutes. The cell pellet was washed once with PBS 1× and suspended in RPMI 1640 medium (Gibco) supplemented with 2 mM L-glutamine (Invitrogen), 100 IU / mL penicillin (Invitrogen), 100 μg / ml streptomycin (Invitrogen), and 10% heat-inactivated porcine serum (Gibco). PAM was maintained in cell culture at 37°C, 5% CO2, or frozen in FBS 10% DMSO (Sigma-Aldrich) and stored at -150°C.

[1087] Primary porcine fibroblasts: Cultured from 2 cm 2Ear tissue sample pieces are used to establish primary fibroblast culture. Briefly, the tissue is cut into small slices and cultivated overnight at 37°C with PBS containing 0.5% trypsin solution. Cells are filtered through a 40 μm cell strainer (Corning) to discard the remaining tissue pieces and centrifuged at 150 × g for 10 minutes. The supernatant is discarded and the cells are resuspended in complete DMEM supplemented with 10% FBS (HyClone, GE HealthCare), 100 IU / ml penicillin (Invitrogen), 100 μg / ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen) and 50 IU / ml nystatin (Sigma-Aldrich). Primary fibroblasts are seeded in T-flasks and maintained their viability after multiple serial passages. Cell passage is performed by trypsinization following standard protocols.

[1088] Peripheral blood mononuclear cells (PBMC): Porcine PBMCs were isolated from whole blood using a Histopaque-1077 (Sigma-Aldrich) density gradient solution. Blood samples drawn from the porcine jugular vein into 10 ml EDTA vacuum tubes (Becton Dickinson) were diluted 1:1 in PBS. The diluted blood was gently layered on top of 10 ml of Histopaque-1077 in a 50 ml conical tube and centrifuged at 400 × g for 30 minutes at 20°C without acceleration or interruption. The whitish layer of blood cells that formed in the interphase containing mononuclear cells was aspirated and transferred to a clean 15 ml conical tube filled with PBS and centrifuged at 400 × g for 10 minutes at 20°C. The supernatant was discarded, and the red blood cells were lysed by performing a hypotonic shock with 9 ml of sterile distilled water for 30 seconds, followed by the addition of 3.5 mL of 3.5% NaCl solution. Afterwards, the cells were centrifuged at 400 × g for 10 minutes at 20°C, washed with PBS, and suspended in RPMI 1640 medium (Gibco) supplemented with 2 mM L-glutamine (Invitrogen), 100 IU / mL penicillin (Invitrogen), 100 μg / ml streptomycin (Invitrogen), and 10% heat-inactivated FBS (HyClone, GE Healthcare). For use in ELISpot analysis, 50 μM β-mercaptoethanol (Sigma-Aldrich) was added to the medium to help maintain a reducing environment.

[1089] Rabbit kidney RK13 cells: Rabbit kidney epithelial RK13 cell line (ATCC CCL-37) was cultured in DMEM supplemented with 10% FBS (HyClone, GE Healthcare), 100 IU / ml penicillin (Invitrogen), 100 μg / ml streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen) at 37°C and 5% CO2.

[1090] African swine fever virus: Two different virulent field isolates of ASFV were used: BA71 (Rodriguez JM et al., PLoS One 2015; 10(11):e0142889; obtained from the spleen of an infected animal in Badajoz, Spain in 1971; GenBank accession number KP055815) and Georgia 2007 / 1 (Chapman DA et al., Emerg Infect Dis. 2011, 17(4):599-605; obtained from a pig tissue sample submitted to the World Organisation for Animal Health Reference Laboratory, Institute for Animal Health, Pirbright, UK on June 4, 2007; GenBank accession number FR682468). The live attenuated BA71ΔCD2 virus, a deletion mutant of BA71 lacking the CD2v gene (EP402R), was previously obtained (WO 2015 / 091322).

[1091] Multi-parameter in silico prediction of CD8+ T cell epitopes: The Georgia 2007 / 1 proteome (UP000141072) was retrieved from Uniprot for in silico CD8+ T cell epitope prediction. Predictions were made using NetMHCpan 3.0 software. Forty-two SLA class I porcine leukocyte antigen (SLA) alleles were considered, and peptides ranging from 8 to 11 amino acid residues with an IC50 (peptide concentration that inhibits 50% of standard peptide binding) below 500 nM were selected. 8,648 different sequences were obtained. To further select the most promising theoretical CTL candidates, additional characteristics of each peptide were evaluated, including:

[1092] i) Proteasomal cleavage was analyzed using the MHC-I processing tool from IEDB (http: / / tools.iedb.org / processing). This program allows the assessment of how effectively a peptide or its N-terminally extended precursor can be released from its source protein by the immunoproteasome.

[1093] ii). Promiscuity: The number of SLA I alleles predicted to bind the peptide with an affinity of 500 nM or less.

[1094] iii). Overlap: The number of predicted peptides with an SLA binding affinity of 500 nM or less that overlap with a given polypeptide in at least one amino acid.

[1095] iv). Peptide immunogenicity: prediction of peptide immunogenicity taking into account its amino acid characteristics and its position within the sequence (Calis J et al., 2013).

[1096] The value of each characteristic was assigned in 10 intervals so that the best value received a score of 10 and the worst value received a score of 1. The final score was expressed as the sum of all values ​​and was finally used to select the best candidate.

[1097] To compare the repertoire of selected peptides, an additional list of TAPREG scores as a novel parameter, previously used to identify CD2v CTL peptides from E75 ASFV strains (Argilaguet et al., 2012), was produced. The TAPREG server uses support vector machine regression to calculate the binding affinity of peptides to TAP. TAPREG scoring newly provides an alternative list, and combines the two peptide lists to obtain the final peptide selection. When overlapping peptides are found in the two lists, the most preferred score and the larger peptide are selected. In addition, larger peptides (15-27 amino acids) are selected based on the presence of more than 10 overlapping peptides in a given hot spot.

[1098] Mass spectrometry-based immunopeptidomics

[1099] PAM infection with ASFV in vitro: 5 × 10 6PAMs / well were infected with ASFV using the specified multiplicity of infection (MOI). Each viral inoculum (0.5 ml) was diluted in complete RPMI without serum and applied to the PAM monolayer. After incubation for 2 hours at 37°C and 5% CO₂, the inoculum was discarded and the cells were fed with complete RPMI supplemented with 10% porcine serum (Gibco). A parallel plate subjected to the same conditions was used to monitor ASFV infection. In this case, cell supernatants were harvested and viral kinetics were analyzed by qPCR as previously described (Lacasta et al., 2014). Cells were incubated at 37°C and 5% CO₂ and harvested by scraping when a cytopathic effect was evident (depending on the MOI used). PAMs were centrifuged at 350 × g for 5 minutes at 4°C and washed with PBS. The supernatant was discarded and the pellets were frozen at -80°C until use.

[1100] Affinity purification of SLA I molecules: SLA I-peptide complexes were immunoprecipitated using 4B7 / 8 α-SLA I antibody-bound CNBr agarose beads (GE Healthcare). Hybridoma culture supernatants harboring mAb α-SLA I were used. Coupling of the antibody to CNBr-activated agarose was performed according to the manufacturer's instructions. The antibody-containing supernatant was dialyzed against 0.5 M NaCl containing 0.1 M sodium carbonate buffer, pH 8.3, at 4°C using a Max D-tube dialyzer (Novagen) with a molecular weight cutoff of 12-14 kDa. Lyophilized agarose was suspended in 1 mM HCl, pH 3, and incubated for 20 minutes at room temperature with end-over-end rotation to wash off lyophilization additives, centrifuged at 500 × g for 2 minutes at room temperature, and washed once with coupling buffer. The antibody-containing coupling solution was added to the washed agarose at an optimal coupling concentration of 0.8-1.2 mg / ml and rotated end-over-end at 4°C overnight. Before and after coupling, measure the OD of the antibody solution at 280 nm to determine the coupling efficiency and, if necessary, incubate for a longer time. The agarose was briefly centrifuged at 500 × g for 2 minutes at RT and the coupling buffer was discarded. Any remaining active groups were blocked with 0.1M Tris-HCl pH 8 for 2 hours at 4°C in a bottom-cap flip rotation. The antibody-coupled agarose was washed using three alternating pH cycles of 0.5M NaCl (acidic wash buffer) containing 0.1M acetic acid pH 4 and 0.5M NaCl (alkaline wash buffer) containing 0.1M Tris-HCl pH 8. Finally, the coupled agarose was resuspended in 150mM NaCl (immunoprecipitation buffer) containing 50mM Tris-HCl pH 8 for immunoprecipitation. PBS 0.1% (w / v) sodium azide was used for long-term storage of the coupled agarose at 4°C.

[1101] Cell pellets were thawed on ice and lysed with 500 μl of 1% n-dodecyl β-D-maltoside (ThermoFisher Scientific) in immunoprecipitation buffer and 1× complete protease inhibitor cocktail (Thermo Fisher Scientific) and incubated at 4°C with end-over-end rotation for 8 hours. The cell lysate was clarified by centrifugation at 20,000 × g for 20 minutes at 4°C and incubated at 4°C with end-over-end rotation for 2 hours with the agarose unlinked to the antibody to remove any proteins that nonspecifically interacted with the agarose. Subsequently, 500 μl of the clarified lysate was added to an equal volume of 4B7 / 8 α-SLA I antibody-conjugated CNBr agarose in immunoprecipitation buffer (approximately 250 μl of agarose in 250 μl of buffer) and incubated at 4°C with end-over-end rotation overnight. Nonspecifically bound molecules were removed by washing with 15-20 agarose volumes of 150 mM NaCl and 50 mM ammonium bicarbonate. The SLA I-peptide complex was eluted in 4-5 agarose volumes of 50% acetonitrile, 5% formic acid and stored at -80°C until analysis.

[1102] Immunoblotting for detection of immunoprecipitated SLA I-peptide complexes: 5% of each sample volume was evaporated to dryness using a Concentrator 5301 (Eppendorf), suspended in 25 μl of 1× NuPAGE LDS sample buffer (Invitrogen) containing 10% β-mercaptoethanol, and heated at 100°C for 5 minutes. Half the sample volume (2.5% of the total elution volume) was run in a 4%-12% gradient NuPAGE Bis-Tris acrylamide SDS-PAGE (Invitrogen) over 1.5 hours in 1× NuPAGE MES SDS running buffer (Invitrogen) containing NuPAGE antioxidant (Thermofisher) at 200 V. A His-tagged protein ladder (Thermofisher) was used as a molecular weight marker. The gel was transferred to a nitrocellulose membrane (Amersham, Protran Premium) using an XCell SureLock™ microcell with a blotting module (Thermofisher) at 50 V for 4 hours in a transfer buffer consisting of 96 mM glycine containing 12 mM Tris-HCl (pH 8) and 20% (v / v) methanol. After transfer, the nitrocellulose membrane was stained with ATX Ponceau S Red Staining Solution (Biochemika Fluka) and destained in distilled water to confirm protein transfer. The nitrocellulose membrane was then blocked in 3% (w / v) skim milk dissolved in wash buffer (TBS 0.1% Tween-20) for 1 hour at room temperature on an orbital shaker with gentle agitation. 4B7 / 8 α-SLA I antibody was added to the membrane at a concentration of 4 μg / ml in blocking buffer and, after washing three times for 20 minutes with wash buffer, incubated for 1 hour at room temperature with gentle agitation. The membrane was then incubated with conjugated anti-mouse IgG HRP (Sigma-Aldrich) diluted 1:10,000 in blocking buffer for 1 hour at room temperature with agitation. For His-tag labeling, conjugated mouse anti-His-tag HRP (Novex) 1:100,000 was used. After thorough washing as described above, the specific signal on the membrane was developed using Western Lightning Ultra chemiluminescent substrate (PerkinElmer) at room temperature in the dark for 5 minutes. Fluorchem HD2 (Alpha Innotech) was used for imaging.

[1103] Tip Desalting and LC-MS / MS Analysis: Samples were desalted using TopTips C18 (PolyLC Inc.) following standard procedures. The eluate from the desalting process was evaporated to dryness and reconstituted in 20 ml of 5% MeOH, 1% HCOOH for analysis by liquid chromatography-mass spectrometry (LC-MS / MS). The MS system used was an LTQ XL Orbitrap (ThermoFisher) equipped with a nanoESI ion source. A total amount (20 μl) of each sample was loaded onto the chromatography system contained in a C18 preconcentrator cartridge (Agilent Technologies) connected to a 15 cm long, 100 μm inner diameter C18 column (Nikkyo Technos Co Ltd). Separation was performed at 0.4 μL / min using a 120-minute acetonitrile gradient from 3% to 40% (Solvent A: 0.1% formic acid, Solvent B: acetonitrile 0.1% formic acid). The HPLC system consists of an Agilent 1200 capillary nanopump, a binary pump, a thermostatic microinjector, and a micro-switching valve. The LTQXL Orbitrap was operated in positive ion mode with a 1.8 kV spray voltage. Spectral analysis was performed in data-dependent mode, acquiring a full scan followed by 10 MS / MS scans of the 10 strongest signals detected in the MS scan of the overall list. Full MS (range 400-1800) was acquired in the Orbitrap with a resolution of 60,000. MS / MS spectra were performed in linear ion trapping.

[1104] Database Search and Peptide Identification: Using SEQUEST (Proteome Discoverer version 1.4, ThermoFisher), all LC-MS / MS spectra were searched using a combined database including wild boar, BA71, and Georgia 2007 / 1 ASFV, and a 6-frame translation of each viral genome was performed (to identify peptides within and outside of known ORFs). The following parameters were fixed: peptide confidence = high, peptide rank = 1, Xcorr>2. In addition, using the identified porcine-specific 9-mer, WebLogo was used to generate sequence logos for each PAM batch. Each logo consists of a stack of symbols, one for each position in the sequence. The total height of the stack indicates the sequence conservation at that position, while the height of the symbol within the stack indicates the relative frequency of each amino acid at that position. The binding site description given by the sequence logo was used to select or eliminate uncertain sequences.

[1105] ASFV gene expression plasmid

[1106] Plasmids encoding full-length ASFV proteins: The ASFV gene expression library used was constructed based on the E75 ASFV isolate (GenBank accession number FN557520.1). The E75 ORF was cloned in-frame with ubiquitin into the pCMV-Ub plasmid (Rodriguez F et al., 2001). An additional plasmid based on the Georgia2007 / 1 sequence (GenBank accession number FR682468) was constructed following the same strategy. A FLAG tag sequence was added before the stop codon of the Georgia2007 / 1 gene to confirm protein expression by immunofluorescence.

[1107] Anti-FLAG tag immunofluorescence to check protein expression: Protein expression of the Georgia 2007 / 1 plasmid was checked in transfected RK13 cells by anti-FLAG tag immunofluorescence. RK13 cells were transfected using the Lipofectamine 3000 transfection kit (Invitrogen) according to the manufacturer's instructions. Mock-transfected cells served as a negative control. After incubation for 2 days at 37°C and 5% CO2, cells were fixed with 3% PFA for 1 hour at 4°C and then permeabilized with PBS containing 0.2% Tween20 for 30 minutes at 37°C. AlexaFluor 488-conjugated anti-FLAG tag monoclonal antibody (MA1-142-A488, Invitrogen) was diluted 1:100 at room temperature and added to the cells for 1 hour. Hoechst 33342 (Life Technologies) was used to stain the nuclei. Finally, cells were examined by fluorescence microscopy.

[1108] Plasmids encoding ASFV multi-epitopes: CTL epitope prediction for the ASFV Georgia 2007 / 1 proteins selected for inclusion in the multi-epitope construct was performed using NetMHCpan 3.0 software. Protein sequences were retrieved from the Georgia 2007 / 1 proteome (Uniprot accession number UP000141072). The 42 SLA I alleles available in NetMHCpan 3.0 were considered, and peptides ranging from 8 to 11 amino acid residues with an IC50 below 500 nM were selected for further analysis. Protein regions containing a high density of predicted epitopes were selected. In Multi-epitope-II (ME-II), each domain also included peptides identified by the aforementioned MS-based immunopeptidomics analysis. A single DNA construct was designed with domains linked by a preferred proteasomal cleavage site (AAY) (Velders et al., 2001) and utilizing ubiquitin as a leader sequence to enhance its SLAI processing and presentation (Rodriguez and Whitton, 2000; Argilaguet et al., 2012; Lacasta et al., 2014). Plasmids encoding multiple epitopes were synthesized by GenScript (New Jersey, USA; SEQ ID NOs: 855 and 856).

[1109] In vivo experiments

[1110] Animals and Animal Safety: Male Blue Rye x Large White piglets were used in all described in vivo experiments. Pigs were fed ad libitum and identified by numbered ear tags, and a seven-day acclimatization period was established before animal manipulation. Animal care and procedures were performed according to Good Experimental Practices guidelines and under the supervision of the Ethical and Animal Welfare Committee of the Universitat Autònoma de Barcelona, ​​Spain.

[1111] Peptide Immunization: Peptide immunization experiments were conducted using three- to four-week-old piglets at the IRTA Monells porcine experimental farm (Girona, Spain). Pigs received two intramuscular tetrad administrations three weeks apart. The peptide cocktail (1 ml) consisted of 20 nM of each peptide in complete Freund's adjuvant (ThermoFisher Scientific) for the first immunization and incomplete Freund's adjuvant (Thermo Fisher Scientific) for the second immunization. EDTA-blood samples were drawn from the jugular vein two weeks after the second peptide administration for PBMC isolation.

[1112] Source of PBMCs to be used as effector cells in various analyses aimed at quantifying ASFV-specific T cell responses: Pigs experimentally infected with Georgia 2007 / 1 produced 100% mortality before they were able to induce ASFV-specific T cells. Therefore, an alternative approach was followed to obtain ASFV-specific T cells. To isolate PBMCs from ASF-convalescent animals, an in vivo BA71ΔCD2 vaccination-Georgia 2007 / 1 challenge experiment was performed at the Biosafety Level 3 facility of the Animal Health Research Center (IRTA-CReSA, Barcelona, ​​Spain). Six to eight-week-old piglets were used, and animals were administered intramuscularly with 10 6 plaque-forming units (PFU) or 3.3×10 4 Three weeks later, the cells were inoculated with a lethal dose of 10 PFU of BA71ΔCD2 in 1 ml of PBS (Monteagudo et al., 2017). 3 Pigs were challenged intramuscularly with 100 genome equivalent copies (GECs) of BA71ΔCD2. Three weeks after BA71ΔCD2 vaccination (before Georgia 2007 / 1 challenge) and two to three weeks after challenge, EDTA-blood samples were drawn and isolated PBMCs were used in various analyses to quantify ASFV-specific T cell responses.

[1113] Heterologous DNA prime-BA71ΔCD2 boost and Georgia 2007 / 1 challenge: The high virulence of Georgia 2007 / 1 precluded the discovery of a lethal challenge dose that would allow pigs to survive for more than a week, thus hindering monitoring of immune responses. To increase the chances of exposure to specific ASFV antigens with protective potential, a prime-boost protocol was established in which animals were primed with a DNA vaccine (encoding specific ASFV antigens) and boosted with a low dose of BA71ΔCD2. Three- to four-week-old male Blue Rhys x Large White piglets were housed together in experimental chambers (12 m) at the IRTA-CReSA biosafety level 3 facility in Barcelona, ​​Spain. 2 ). Pigs were immunized two weeks apart with two doses of 0.6 mg of the corresponding endotoxin-free DNA plasmid or plasmid mixture (Qiagen) in 1.5 ml of saline. According to the previously described optimization protocol, one-third of each vaccine dose was injected intramuscularly into the quadriceps femoris, one-third was injected intramuscularly into the trapezius muscle of the neck, and the last third was injected subcutaneously into the ear (Argilaguet et al., 2011). Following the same administration schedule, control pigs received the empty pCMV-Ub plasmid. Two weeks after the second DNA dose, the suboptimal (partially protective) dose of 10 3 Three weeks later, the pigs were given a lethal dose of 10 PFU of the Georgia 2007 / 1 ASFV isolate. 3 Pigs were challenged intramuscularly with GECs. Blood samples were drawn from the jugular vein, and nasal swabs were collected before and after each of the following time points: DNA prime (0, 4, and 7 days per week), BA71ΔCD2 boost (0, 4, 7, and 14 days per week), and Georgia 2007 / 1 challenge (0, 4, 7, 14, and 21 days per week). Necropsies were performed to confirm or abrogate the presence of ASFV-compatible pathological lesions.

[1114] Monitoring for ASF-compatible clinical signs: Animals were observed daily according to a welfare schedule to monitor their health and record clinical signs following infection with ASFV. Clinical assessments included rectal temperature, behavior, body condition (vertebral and rib hump), cyanosis, gastrointestinal symptoms, and respiratory symptoms. Each parameter was scored on a scale of 0 to 3 (0: normal, 1: mild, 2: moderate, 3: severe) based on severity, as described by Galindo-Cardiel et al. A humane endpoint was reached when disease progression resulted in an unacceptable loss of overall welfare (Galindo-Cardiel et al., 2013).

[1115] Quantification of viral titers in serum and nasal swabs by qPCR: Viral DNA from serum and nasal swab-PBS suspensions was quantified using the SYBR Green real-time PCR (qPCR) method described previously (Lacasta et al., 2014). Briefly, viral genomic DNA was obtained from 200 μl of serum or swab-PBS suspension using the NucleoSpin blood kit (Macherey-Nagel) and then used as a template for amplification of an 85 bp long fragment from the ASFV serine protein kinase gene (R298L) using PowerUp SYBR Green master mix (Thermo Fisher Scientific). Results are expressed as log 10 GEC number / ml serum or nasal swab, and in 10 3 The detection limit of the assay was established at 10 GEC / ml.

[1116] Immunological readouts

[1117] Porcine IFNγ ELISpot: IFNγ responses were assessed by ELISpot analysis using purified mouse anti-porcine IFNγ clone P2G10 (BD Pharmingen) as the capture antibody and biotinylated mouse anti-porcine IFNγ antibody P2C11 (BD Pharmingen) as the detection antibody, following previously reported methods (Lacasta et al., 2014). 96-well plates (Costar 3590, Corning) were coated with 5 μg / ml capture antibody in carbonate-bicarbonate buffer, pH 9.6, at 4°C overnight. Plates were washed three times with PBS and blocked with complete RPMI with 10% FBS for 1 hour at 37°C. 5×10 cells were used in the presence of the corresponding stimuli. 5 PBMC / well, final volume 200μl. Peptide was added as stimulator at a final concentration of 4μg / ml, and RPMI and 10μg / ml phytohemagglutinin-M (PHA-M, Sigma-Aldrich) were used as negative and positive controls, respectively. When LAV BA71ΔCD2 was used as stimulator, 10 5PFU. After incubation at 37 ° C, 5% CO2 overnight, the cells were washed with PBS 0.05% Tween20, and IFNγ was detected using 0.5 μg / ml biotinylated anti-porcine IFNγ antibody at 37 ° C for 1 hour. After washing, ELISpot was displayed by adding 50 μl of insoluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Calbiochem) and stopped by washing with water. The frequency of specific IFNγ-secreting cells (IFNγ-SC) shown in the figure is the average of two replicates minus the counts in the negative control wells. 300 spots / well is considered the limit of analytical resolution (wells with more than 300 spots receive a score of 300).

[1118] For the use of fibroblasts as APCs in ELISpot analysis, the ratio used was 1 APC: 5 autologous PBMCs. Plasmid transfection of fibroblasts was performed by electroporation using the Neon transfection system 10 μl kit (Invitrogen). Fibroblasts were collected by trypsinization, centrifuged at 250 × g for 5 minutes at RT and washed with PBS. An appropriate number of cells (100,000 fibroblasts / condition) were placed in a clean Eppendorf tube and suspended in 10 μl Neon resuspension buffer R and mixed with 500 ng of the corresponding plasmid or plasmid mixture. Electroporation was performed under the following pulse conditions: pulse voltage = 1700 V, pulse width = 20 ms, number of pulses = 1. Fibroblasts electroporated with an empty pCMV-Ub plasmid were used as a negative control. Finally, the electroporated cells were placed in the corresponding wells of a 96-well plate together with autologous PBMCs and proceeded as described above. When transfected fibroblasts were treated, no replicates were made. The number of spots in control wells with fibroblasts transfected with the empty pCMV-Ub plasmid, which never exceeded 10, was subtracted from the specific IFNγ-SC indicated in the figure.

[1119] Detection of ASFV-specific antibodies by ELISA: ASFV-specific antibodies were detected in pig serum using an OIE-approved indirect ELISA assay based on the use of soluble extracts from ASFV-infected cells (Gallardo et al., 2013). Positive sera were detected using a 1 / 20,000 dilution of peroxidase-conjugated anti-swine IgG (Sigma-Aldrich) as a secondary antibody and soluble TMB as a specific peroxidase substrate (Sigma-Aldrich). The reaction was stopped with 1N HSO (Sigma-Aldrich). Plates were read at a wavelength of 450 nm and results expressed as optical density (OD) values.

[1120] Example 2 - Computer simulation prediction, immune peptide omics and gene library: identification of ASFV CD8+ T cell epitopes

[1121] This study aimed to investigate the effectiveness of three different strategies for identifying ASFV CD8+ T cell epitopes with protective potential against the Georgia 2007 / 1 ASFV isolate, currently circulating in continental Europe and China.

[1122] The first approach explored here was a multiparameter bioinformatics analysis using the Georgia 2007 / 1 proteome as a template for predicting peptide sequences that are more likely to be promiscuously represented by the SLA I pathway.

[1123] The second strategy employed here was to characterize the repertoire of ASFV SLA I-binding peptides found in PAMs infected with the virus in vitro. The potential of individual peptides, derived from both in silico predictions and immunopeptidomics analysis, to stimulate ASFV-specific T cells was assessed by IFNγ ELISPOT using PBMCs from animals vaccinated with the LAV BA71ΔCD2 as effector cells. Because BA71ΔCD2 conferred protection against the heterologous Georgia 2007 / 1 strain, it was hypothesized that protective antigens were shared between the two isolates. In this in vitro stimulation assay, the peptides directly bound to SLA I molecules exposed on the cell surface and were able to stimulate specific CD8+ T cells, albeit limited by their specific SLA I molecule match.

[1124] The third strategy tested here attempted to overcome the haplotype specificity of peptide-based assays by using full-length proteins that potentially contain epitopes with multiple SLA I specificities as in vitro stimulators. To enhance SLA I processing and antigen presentation, gene expression plasmids encoding separate full-length ASFV ORFs fused to ubiquitin (Rodriguez and Whitton, 2000; Rodriguez et al., 2001) were used as sources of ASFV antigens for analysis. The individual plasmids were transfected into porcine skin fibroblasts, which then served as APCs in an IFNγ ELISPOT assay using autologous PBMCs from ASF-convalescent pigs as effector cells.

[1125] result

[1126] Evaluation of Georgia 2007 / 1 CD8+ T cell epitope predictions: Sequences that scored the most optimal theoretical ranking in a multiparameter bioinformatics analysis using the Georgia 2007 / 1 proteome as a template were synthesized. The final selected set included 330 peptides from 110 ASFV proteins. 266 peptides were direct outputs of the prediction software and therefore ranged in length from 8 to 11 amino acids. Due to the presence of multiple peptides with 10 or more overlapping predictions, 64 longer sequences (12-27 amino acids in length) were selected.

[1127] Of the 330 predicted peptides, only one induced an IFNγ response in PBMCs from Georgia 2007 / 1 survivors previously immunized with BA71ΔCD2, yielding a 0.3% predicted peptide in silico. The immunogenic peptide corresponds to residues 68-86 of MGF100-1L (SEQ ID NOs: 570 and 571) and showed specific IFNγ secretion in 11 of 20 animals tested (55%). While the peptide is a 19-mer and therefore not a direct result of the software used, it was selected because the peptide within that sequence overlaps with more than nine predicted CD8+ T cell epitopes.

[1128] Evaluation of SLA I-Defined Peptides Identified by Mass Spectrometry-Based Immunopeptidomics: MS-based immunopeptidomics analysis was performed using PAMs infected with Georgia 2007 / 1, BA71, or LAV BA71ΔCD2. Viral replication in the cells was confirmed by increasing viral titers in the supernatant as assessed by qPCR. Following anti-SLA I immunoprecipitation and elution, the presence of SLA I-peptide complexes was confirmed by immunoblotting. Bands located between 40 kDa and 50 kDa are consistent with the expected molecular weight of approximately 45 kDa for class I SLA heavy chains. The slightly heavier band and the 25 kDa band most likely correspond to the heavy and light chains of the anti-SLA I antibody used for immunoprecipitation, which had been separated from the agarose beads. Samples from uninfected PAMs were also analyzed by immunoblotting to eliminate the possibility of nonspecific interactions of the anti-SLA I antibody.

[1129] Unfortunately, no peptides were found in macrophages infected with Georgia 2007 / 1, regardless of the PAM used or the multiplicity and duration of infection. In contrast, macrophages infected with BA71 or BA71ΔCD2 conferred SLA I-specific peptides. These comparative analyses confirmed that the absence of Georgia 2007 / 1 SLA I-defining peptides was strain-specific.

[1130] Using PAMs from three animals, 135 SLA I-binding peptides (106 different sequences) from 56 different ASFV proteins were identified. For both BA71 and Georgia 2007 / 1 isolates, 84.3% of the identified sequences were identical, 13% of the peptides differed by only one amino acid, which theoretically does not play a critical role in SLA I binding, and only 1.7% of the peptides showed considerable divergence in their sequences between the two viruses, thus confirming the usefulness of this method for identifying highly conserved peptides between ASFV strains.

[1131] Interestingly, while BA71-infected PAMs resulted in 44 ASFV sequences, 88 peptides were generated from samples infected with BA71ΔCD2. As expected for a SLAI ligand, peptide lengths ranged from 8 to 13 amino acids, with 50% of peptides being 9-mers. From a functional perspective, the largest percentage of peptides was for proteins of unknown function, accounting for 35.6% of the total peptides, but peptides from proteins involved in transcription and replication, morphogenesis, and host-cell interactions, as well as from multigene families, were also identified. Regarding transient expression of proteins during the infectious cycle, early, intermediate, and late proteins were identified, with the latter accounting for the highest percentage (35.6%), but 45.9% of the peptides were from proteins with unknown transient expression.

[1132] Of the nine peptides, the ASFV protein from which a significant number of peptides were identified was the atypical protein B475L, followed by the structural polyprotein pp220 encoded by the CP2475L gene and the helicase encoded by the D1133L ORF, from which eight and seven peptides were detected, respectively. Furthermore, five SLAI peptides were located in regions outside of any known ORF, confirming previously described results (Jenson et al., 2000), and some of these SLAI peptides do not even have a conventional start codon. Surprisingly, all five out-of-frame peptides were identified from samples infected with BA71ΔCD2. This supports the idea that these out-of-frame peptides may also be expressed and play a role in triggering protective responses against the Georgia 2007 / 1 ASFV, as homologous sequences are found in the genome of the Georgia 2007 / 1 isolate.

[1133] Of the 111 different peptides identified by the immunopeptidomics approach, 5 peptides induced IFNγ responses in PBMCs from animals surviving the Georgia 2007 / 1 challenge (Table 1), representing 4.5% of the total peptide count. Interestingly, three peptides recognized by more than one animal tested were identified in PAMs infected with BA71ΔCD2, whereas antigenic peptides derived exclusively from BA71-infected samples induced IFNγ responses in only 10% of animals. This is by no means conclusive: it suggests that the peptide repertoires of BA71ΔCD2 and BA71 are slightly different.

[1134] Table 1. ASFV epitopes from immunopeptidomics studies induce IFNγ responses in vitro in PBMCs from ASF convalescent animals vaccinated with LAV BA71ΔCD2. Animals were classified as responders if 20 or more spots were counted.

[1135]

[1136] As expected, the peptides were not uniformly recognized by all pigs, most likely reflecting their apparent restriction to specific SLA alleles. Supporting this idea, vaccination of pigs with a Freund's adjuvant cocktail containing approximately 25 peptides identified by MS-based immunopeptidomics showed that some of the peptides were immunogenic, but again not consistently recognized by all pigs (Table 2). The peptides used here were identified in the first immunopeptidomics analysis performed. Two vaccination groups were defined based on the theoretical binding affinity of each peptide to the SLA I allele available at NetMHCpan3.0. Peptides with high binding affinity (IC50 < 1000 nM) for most of the alleles analyzed were classified as strong binders, while peptides with lower theoretical binding affinity (IC50 > 1000 nM) were grouped as weak binders. Two identified peptides from proteins D1133L and I226R were classified as strong binders, while the third identified peptide from protein G1211R was a theoretical weak binder. Notably, contrary to expectations based on theoretical predictions, the theoretical weak binders were recognized by 66.7% of the animals tested, while the two strong binders induced an IFNγ response in only one of six pigs (Table 2).

[1137] Table 2 - ASFV-specific epitopes induce IFNγ responses assessed by ELISpot in PBMCs of animals vaccinated with peptide cocktail in Freund's adjuvant.

[1138]

[1139] Use of gene expression plasmids to identify immunodominant ASFV CD8+ T cell antigens: As reflected above, peptide-based approaches exhibit a major drawback: the restricted representation of defined SLA haplotypes.

[1140] With the goal of avoiding this limitation and attempting to extend the studies described above, we aimed to identify promiscuous CD8+ T cell determinants from ASFV, focusing on its full-length antigen. The pCMV-Ub plasmid encoding the full-length ASFV ORF was transfected into primary fibroblasts, and these transfected fibroblasts were used as APCs in an ELISpot assay using PBMCs (autologous) from the same animal as effector cells.

[1141] The most preferred conditions for electroporation of fibroblasts using the Neon transfection system were previously set up using different electroporation configurations to transfect pCMV-GFP plasmids into primary porcine fibroblasts. The most preferred conditions were selected based on the percentage of transfected cells (GFP+) relative to viable cells. The conditions used gave 36.14% GFP+ cells and a 4.60% mortality rate. Although these values ​​may not be guaranteed to be constant in the following analysis, they serve as a proof of concept for demonstrating that primary fibroblasts can express proteins with the help of the pCMV promoter.

[1142] A collection of 73 recombinant plasmids from the ASFV gene expression library available as described above was used. Although the gene expression library contains nearly 100 recombinant plasmids, the 15 recombinant plasmids tested and other recombinant plasmids encoding known immunogenic proteins were not included here. In the first screening step, a mixture of 10 or 11 plasmids was electroporated into fibroblasts to subsequently test individual plasmids from the mixture for their ability to specifically induce an IFNγ response. Expression of similar ASFV gene expression plasmids was confirmed by adding a FLAG tag sequence to the C-terminus of the ASFV gene and detecting it by immunofluorescence as described in this and the following examples.

[1143] One single clone was identified from the ASFV gene expression library that was able to specifically stimulate IFNγ expression in all animals tested except one: pCMV-Ub-MGF505-7R ( Figure 1 ). Non-responder pigs (pig 13) showed ASFV-specific IFNγ-SC, thus abolishing the possible immunosuppressive state of PBMC. However, it cannot be ruled out that some specific electroporation events (one transfection / plasmid) failed. Notably, a single peptide from MGF505-8R (MGF505-7R334-341: NSTLVIRI; SEQ ID NO: 717) was recognized in the immunopeptidomic analysis using PAM infected with BA71ΔCD2. The fact that this peptide was recognized by a small proportion of ASF convalescent pigs confirms its SLA I-restricted nature and positively suggests the advantage of using the full-length MGF505-7R protein containing multiple CD8+ T cell determinants in future development.

[1144] Interestingly, this approach was used to identify two additional antigenic proteins: A238L and MGF100-1L. Although these two proteins showed lower promiscuity than MGF505-7R, they were still largely recognized by ASFV-specific T cells. Thus, fibroblasts transfected with recombinant plasmids encoding full-length A238L and MGF100-1L induced IFNγ responses in 60% and 50% of animals, respectively, after in vitro stimulation with ASFV. Figure 2Interestingly, peptides previously identified from these proteins by immunopeptidomics analysis: A238L81-91 and MGF100-1L68-86, specifically stimulated IFNγ responses in 30% and 50% of pigs, respectively.

[1145] Conclusion: The results presented above strongly suggest that immunopeptidomic analysis of PAMs infected with ASFV in vitro is a more reliable strategy than in silico predictions for identifying ASFV-specific CD8+ T cell epitopes. However, the peptides were not uniformly recognized by all pigs, likely reflecting a significant restriction to specific SLA alleles. This was overcome by using full-length proteins, which were used here to identify MGF505-7R as a novel immunodominant and promiscuous ASFV antigen, as well as two additional antigens (i.e., A238L and MGF100-1L, which were recognized by at least 50% of the animals tested). Therefore, focusing on full-length proteins rather than epitopes may be a more suitable approach for identifying ASFV antigens with the potential to promiscuously induce specific T cell responses.

[1146] Example 3 - M448R and MGF505-7R: Two immunodominant ASFV antigens with protective potential

[1147] Since the significant restriction of peptides to specific SLA alleles is one of the major drawbacks of peptide-based vaccination approaches, the present study focused on full-length proteins. Based on the identification of SLA I-restricted peptides by MS-based immunopeptidomics, 15 ASFV-encoded proteins were selected as potential inducers of CD8+ T cell responses.

[1148] To assess their immunogenicity and protective potential, pigs were vaccinated with 15 selected recombinant plasmids, each encoding an ASFV antigen with a ubiquitin sequence at its N-terminus, designed to optimize SLA I presentation and enhance the induction of CD8+ T cell responses. As explained in Example 1, a heterologous regimen was employed, consisting of a DNA-primed immunization with the 15 selected antigens followed by a low-dose vaccination with live, attenuated BA71ΔCD2 ASFV. The cross-protective capacity of BA71ΔCD2, which confers protection not only against the parental BA71 but also against heterologous viruses, including the Georgia 2007 / 1 isolate, allows for the enhancement of any cross-protective responses induced by the recombinant plasmids, thereby increasing the chances of identifying relevant antigen-specific T cells. Furthermore, this vaccination regimen allowed for the assessment of the ability of pigs primed with the 15 selected antigens to achieve protection against a lethal challenge with Georgia 2007 / 1, compared to a control group primed with a plasmid that does not encode any ASFV-specific proteins.

[1149] In the first experiment described here, partial protection against Georgia 2007 / 1 challenge was observed in pigs that received 15 selected antigens as a DNA prime. Among the antigens included in the plasmid cocktail, one protein, M448R, displayed the highest immunodominant properties. Furthermore, M448R exhibited a promiscuous cellular response in ASFV-convalescent pigs that did not receive a DNA prime. As described in Example 1, the use of fibroblasts transiently expressing ASFV antigens as APCs for autologous PBMCs from ASF-convalescent pigs allowed the recognition of another ASFV protein, MGF505-7R, with promiscuous properties similar to those of M448R. Therefore, both M448R and MGF505-7R are promising candidates for further exploration of their importance in protective immunity against ASFV.

[1150] Therefore, the present study describes a second in vivo experiment designed to evaluate the immunogenicity and protective potential of the combined M448R and MGF505-7R using the prime-boost vaccination regimen as explained above.

[1151] result

[1152] DNA immunization with a mixture of plasmids encoding 15 pre-selected ASFV proteins confers partial protection against Georgia 2007 / 1 challenge infection: Based on the previous results of SLAI immune peptidomics studies of ASFV-infected PAMs, 15 ASFV antigens were selected as potential candidates for inducing CD8+ T cell responses and evaluated for their immunogenicity and protective potential against Georgia 2007 / 1 lethal challenge.

[1153] The selected panel includes three potential ASFV enzymes potentially involved in nucleic acid metabolism: D339L (RNA polymerase subunit 7), EP424R (putative methyltransferase), and M448R (RNA ligase); as well as the putative transcription factor I243L. Additionally, two proteins involved in virion morphogenesis were included: the structural protein p37, a processing product of the polyprotein pp220, and the chaperone protein B602L. Also selected were the multigene family 505 members MGF505-1R and MGF505-3R, as well as seven proteins of unknown function: B475L, DP238L, H339R, I226R, I73R, I9R, and K145R. All of the above-mentioned proteins represent early, late, and intermediate proteins during the ASFV replication cycle. Expression of the ASFV proteins tested here was confirmed by anti-FLAG tag immunofluorescence in transfected RK13 cells, as described in Example 1.

[1154] Following the vaccination schedule described in Example 1, three of five pigs (60%) primed with 15 recombinant plasmids survived the lethal challenge with Georgia 2007 / 01. In contrast, and consistent with previous results from the laboratory using a low dose of BA71ΔCD2, only one of five control pigs (20%) immunized once with a low dose of BA71ΔCD2 survived ( Figure 3 ).

[1155] During the experiment, animals were monitored daily for typical ASF clinical symptoms, including fever, lethargy, general physical condition, gastrointestinal symptoms, respiratory symptoms, and cyanosis. Although four surviving pigs developed transient ASF-compatible symptoms, the three surviving pigs primed with 15 recombinant plasmids showed milder clinical symptoms compared to surviving pigs from the control group. These results were associated with: i) a delayed and shorter duration of serum viremia in the surviving animals in the group primed with 15 recombinant plasmids, and ii) a 1 to 2 log reduction in the maximum titer of their challenge virus. 10 iii) undetectable virus at any time post-challenge. In addition, a one to two log reduction in nasal shedding was observed compared to both non-surviving and surviving animals in the control group. 10 Decrease.

[1156] The survivor from the control group (pig 185) showed a high and prolonged fever peak (>41°C for 5 days) that began at 4 dpc with lethargic behavior, while the three surviving animals in the group primed with 15 recombinant plasmids had mild fever that lasted for up to three days. Throughout the study, pig 181 experienced sporadic symptoms consistent with mild fever peaks, and pig 184 had no obvious symptoms. However, the surviving pig 180 also showed lethargic behavior and obvious breathing difficulties. The onset of clinical symptoms was delayed compared to both the surviving controls and non-survivors. Pigs 182 and 183 of the "15 clones" group succumbed at 9 dpc and 8 dpc, respectively, and the temperature and viral load in their serum and nasal swabs were similar to those in the serum and nasal swabs of the control group.

[1157] Immunization with 15 recombinant plasmids induced ASFV-specific T cells, but no antibody response was detected: Administration of 15 recombinant plasmids did not induce any specific antibody response, but it induced a detectable ASFV-specific IFNγ response at 14 dpp, thus indicating the immunogenicity of at least one of the 15 included antigens.

[1158] Following BA71ΔCD2 vaccination, all animals seroconverted and developed ASFV-specific T responses. No clear correlation was observed with the levels of antibodies or T cells induced after vaccination, as control animals vaccinated only once with BA71ΔCD2 also displayed significant ASFV-specific immune responses. Nevertheless, two animals from the "15 clones" group that did not survive the Georgia 2007 / 1 challenge displayed the lowest antibody levels and ASFV-specific T responses upon challenge infection. T cell responses induced directly after DNA vaccination likely contributed to the better control of ASFV infection and viral clearance in the "15 clones" primed group.

[1159] M448R showed immunodominant properties in ASFV convalescent animals previously primed with 15 recombinant plasmids: Once the immunogenicity of the administered plasmid cocktail was confirmed, the aim was to determine the immunogenicity profile of each of the 15 ASFV antigens used. For this purpose, porcine fibroblasts were electroporated with each individual recombinant plasmid contained in the immunization mixture and used as APCs in an ELISpot assay using autologous PBMCs obtained at 14 dpc as effector cells.

[1160] Interestingly, when PBMCs were cultured with fibroblasts transfected with pCMV-Ub-M448R, high levels of IFNγ-SC (>50 spots) were exclusively detected in all animals. The number of M448R-specific IFNγ-SCs was comparable to that obtained when transfecting a mixture with 15 recombinant plasmids. This result suggests that T cell immunity against M448R is largely responsible for the immunogenicity observed after immunization with 15 recombinant plasmids. In addition, the M448R-specific T cells primed may contribute to the milder course of Georgia 2007 / 1 infection and increased survival in the "15 clones" group.

[1161] The number of spots was much higher when ASFV was used as a stimulus after the boost than when porcine fibroblasts transfected with the pCMV-Ub-M448R plasmid were used. This is most likely explained by different reasons. First, ASFV infects APCs much better than plasmid-transfected APCs and are therefore processed more efficiently. Second, PBMCs from pigs convalescent with ASFV are also more likely to recognize antigens present in ASFV than those contained in the plasmid mixture.

[1162] M448R induces specific T cell responses during ASFV infection without prior DNA priming: In the next step, we sought to determine whether M448R induces ASFV-specific CD8+ T cells not only when pigs are primed with pCMV-Ub-M448R, but also after ASFV infection. To this end, PBMCs from animals vaccinated with BA71ΔCD2 and challenged with a lethal dose of Georgia 2007 / 1 (without prior pCMV-Ub-M448R plasmid priming) were tested in an ELISpot assay using autologous porcine fibroblasts transfected with pCMV-Ub-M448R plasmid. Surprisingly, IFNγ responses against M448R when expressed in a pCMV-Ub plasmid were induced in 7 of 9 animals, thus confirming the promiscuous nature of M448R and the presence of immunodominant T cell epitopes within it.

[1163] Immunization with pCMV-Ub-M448R and pCMV-Ub-MGF505-7R confers partial protection against lethal challenge with Georgia 2007 / 1: Given the immunodominant nature of M448R and its protective potential, it was decided to include it in future experimental vaccine formulations along with MGF505-7R, a second antigen that is promiscuously recognized by ASF convalescent pigs (Example 1). Therefore, the immunogenicity and protective potential of these two antigens were evaluated by priming a group of pigs with two DNA plasmids encoding the M448R and MGF505-7R proteins with a ubiquitin sequence at the N-terminus and boosting with a low dose of BA71ΔCD2.

[1164] Consistent with the previous results using low doses of BA71ΔCD2, only one of five animals in the control group (20%) survived the Georgia 2007 / 1 challenge infection. In contrast, three of five pigs in the group immunized with pCMV-Ub-M448R+pCMV-Ub-MGF505-7R survived the lethal challenge infection ( Figure 4 ).

[1165] Surviving animals from the DNA-primed group showed lower and shorter fever peaks than control pigs. Thus, pig 89 did not show fever or other clinical symptoms at any time after the Georgia 2007 / 1 challenge, and pig 90 showed a brief fever peak at 20-21 dpc. The third survivor in this group (pig 88) showed mild lethargy and slightly affected body condition (clinical score never exceeded 2), but fully recovered by day 14 post-challenge. Pigs 86 and 87 from the DNA-primed group succumbed to the infection, showing ASF clinical symptoms that were indistinguishable from those found in the control pigs (vaccinated only with BA71ΔCD2). These ASF clinical symptoms included severe ASF symptoms such as lethargy, depression, visible spine and / or ribs, difficulty breathing, and cyanosis (scored at least 4 on the clinical symptom scale).

[1166] Clinical signs of ASF were more pronounced in the control group, with two exceptions: Pig 97 was found to have died unexpectedly late (on 19 dpc) after suffering from mild ASF symptoms and high fever (>41°C) for at least 2 consecutive days before death, and Pig 99, although surviving the Georgia 2007 / 1 challenge, suffered from prolonged lethargy that began on 9 dpc and lasted until the end of the trial, and also suffered from cyanosis of the ears and tail.

[1167] Although the survival percentage in the group primed with M448R+MGF505-7R was the same as when primed with 15 clones (60%), the animals appeared to cope better with the Georgia 2007 / 1 infection, at least based on the clinical symptoms observed.

[1168] Priming with pCMV-Ub-M448R and pCMV-Ub-MGF505-7R helped reduce viral titers in serum and nasal shedding after Georgia 2007 / 1 challenge infection: Serum and nasal swabs were collected on designated sampling days and subsequently tested for the presence of ASFV DNA by qPCR. Following BA71ΔCD2 vaccination, no viral DNA was found in serum or nasal swabs from DNA-primed animals. In contrast, and indicative of LAV replication, a peak in viral DNA was detected in serum in one control animal after BA71ΔCD2 administration.

[1169] After Georgia 2007 / 1 vaccination, pigs that had to be sacrificed (all pigs reached at least 10 at some point after infection) 7 The viral DNA content in the serum of the surviving animals remained below 10 6GECs / ml. Focusing on surviving animals, with the exception of a viral spike in animal 90 at 21 days per week, no ASFV DNA was detected in the serum of animals 89 and 90, and low levels of ASFV DNA were found in nasal swabs. These results are consistent with the absence of reported clinical symptoms in these animals. Detection of ASFV DNA in both serum and nasal swabs from animal 88 at 7 days per week was consistent with the mild ASF symptoms observed in this surviving animal. Despite reports of prolonged lethargy and cyanosis, the surviving animal in the control group (pig 99) was able to control viral replication, demonstrating low serum viral DNA levels and reduced nasal shedding. The severe ASF clinical symptoms observed in animals from the DNA-primed group that had to be sacrificed were consistent with high viral titers in both serum and nasal swabs, and no differences were found between the succumbed animals in the vaccinated and control groups.

[1170] DNA immunization with pCMV-Ub-M448R and pCMV-Ub-MGF505-7R induced ASFV-specific T cell responses that recognized both M488R and MGF505-7R antigens in vitro: As expected, administration of the pCMV-Ub-M448R and pCMV-Ub-MGF505-7R plasmids did not induce any detectable ASFV-specific antibody responses. No differences in antibody responses were observed between surviving and succumbing animals before Georgia 2007 / 1 vaccination, with all animals showing elevated levels on the day of challenge (except pig 100, which also showed a weak T cell response).

[1171] As expected, IFNγ responses to porcine fibroblasts transfected with pCMV-Ub-M448R and pCMV-Ub-MGF505-7R were detected 7 days after the second DNA immunization in DNA-primed animals, but not in the control group. Although low levels of IFNγ-SC were detected (likely due to the low immunogenicity of DNA vaccines in large animals), this confirms the immunogenicity of the tested antigens when administered in a DNA-based formulation. Notably, at this early time point, the two animals (pigs 86 and 87) that did not show M448R and MGF505-7R-specific T cell responses were the ones that later succumbed to the Georgia 2007 / 1 challenge.

[1172] Consistent with previous results demonstrating the promiscuous and immunodominant nature of both M448R and MGF505-7R during ASFV infection, following BA71ΔCD2 vaccination, all animals except one control (pig 100) were able to recognize their autologous porcine fibroblasts transfected with a cocktail of recombinant plasmids containing pCMV-Ub-M448R and pCMV-Ub-MGF505-7R. Throughout the experiment, the non-responsive controls showed low cellular and humoral responses, which likely reflects an immunosuppressive state.

[1173] To characterize the responses induced by each of the antigens tested here, ELISpot analysis was performed using porcine fibroblasts transfected with either pCMV-Ub-M448R or pCMV-Ub-MGF505-7R. Simultaneous IFNγ responses to both M448R and MGF505-7R were detected in all animals (except pig 86, which did not recognize MGF505-7R), thus negating a possible immunodominance effect between M448R and MGF505-7R when administered in a DNA-based formulation. At the group level, the three surviving animals (pigs 88, 89, and 90) had higher IFNγ responses to both M448R and MGF505-7R than the two animals (pigs 86 and 87) that died at all analyzed time points. Reconfirming the presence of M448R- and MGF505-7R-specific T cells in Georgia 2007 / 1 convalescent animals without prior DNA priming, surviving control animals (pig 99) showed significant IFNγ responses to both antigens at the end of the experiment.

[1174] To determine whether DNA priming vaccination had an effect on the magnitude of ASFV-specific T cell responses, the number of IFNγ-secreting cells responding to BA71ΔCD2 was assessed early and late after the boost (7 days and 21 dpb, respectively). At 21 dpb, all pigs showed indistinguishable ASFV-specific T cell responses, with the exception of low-responder pig 100. IFNγ responses were also detected in all animals at 7 dpb, but no significant differences were found between DNA-primed animals and the control group. It is worth noting that the top IFNγ responder, pig 99, within the control group, was the only survivor.

[1175] Conclusion: In this study, we confirmed the feasibility of inducing ASFV-specific cellular responses in pigs by administering a cocktail of 15 recombinant plasmids encoding full-length ASFV proteins in frame with ubiquitin. We demonstrated that a heterologous vaccination regimen consisting of a DNA prime with the 15 recombinant plasmids followed by a low dose of LAV BA71ΔCD2 conferred partial protection against a Georgia 2007 / 1 challenge. Protein M448R was primarily responsible for the immunogenicity of the plasmid cocktail, thus demonstrating its protective potential. Furthermore, ASF convalescent animals that had not received a prior DNA prime recognized M448R promiscuously.

[1176] Following the same experimental design, DNA priming with M448R and MGF505-7R (Example 1), which was also shown to be immunodominant and promiscuous, also resulted in a 60% survival percentage.

[1177] Example 4 - Design of multiple epitope-based DNA constructs and evaluation of their immunogenicity and anti-ASFV protective potential

[1178] In a first attempt to enhance the immunogenicity of DNA constructs based on the Georgia 2007 / 1 sequence, ASFV proteins, including the previously described CD8+ T cell determinants EP402R, CP312R, and A240L, were analyzed for the presence of regions containing multiple theoretical CTL epitopes. These protein regions, or "SLAI hotspots," were selected for inclusion in the vaccine formulation with the goal of inducing a broad repertoire of SLA I-restricted immune responses. Optimal proteasomal cleavage sites were added to separate the different protein domains, and the ubiquitin gene was used as a leader sequence. In this design, the goal was to enhance SLA I processing and epitope presentation as previously reported, thereby inducing specific CD8+ T cell responses while eliminating humoral responses. The immunogenicity of this multi-epitope-encoding plasmid, referred to herein as Multi-Epitope-I (ME-I; SEQ ID NO: 855), was confirmed in vivo. Confirming the efficacy of the strategy, pigs vaccinated with ME-I induced ASFV-specific T cell responses that specifically recognized peptides from EP402R, CP312R, and A240L.

[1179] Extending these results to identify novel Georgia 2007 / 1 antigens, proteins with the potential to induce CD8+ T cell responses were selected according to Example 2. Therefore, selection was made based on in vitro immunopeptidomic profiling of ASFV-infected macrophages, followed by in silico CTL epitope prediction for each of these macrophages. Protein regions containing a high density of predicted epitopes and at least one SLA I-defined peptide identified in the immunopeptidomic analysis were selected as "SLA I hotspots" for the design of a second multi-epitope DNA construct (ME-II; SEQ ID NO: 856).

[1180] To increase the chances of success of the experimental vaccine prototype, a heterologous prime-boost immunization protocol as described in Example 1 was used. Thus, animals were primed with a DNA plasmid encoding a multi-epitope construct and then inoculated intramuscularly with a low dose of live, attenuated BA71ΔCD2 virus. This model was used not only to confirm the ability of the selected antigens to induce ASFV-specific CD8+ T cells, but also to assess their protective potential against a lethal challenge with Georgia 2007 / 1.

[1181] result

[1182] Selection of ASFV proteins with the potential to trigger immunodominant CD8+ T cell responses and design of multi-epitope DNA constructs: The results of the SLAI-defined immunopeptidomic analysis were used to select ASFV proteins with the potential to induce CD8+ T cell responses. The most preferred protein candidates were selected based on three main criteria: (i) proteins from which 5 or more peptides were recognized in the SLAI-defined immunopeptidomic analysis, (ii) proteins from which peptides were recognized using PAMs from different animals, and (iii) proteins from which peptides were recognized by specific T cells obtained from pigs vaccinated with live attenuated virus (LAV) BA71ΔCD2 or a peptide cocktail including that specific peptide (i.e., antigenic peptide).

[1183] With this data in mind, 13 proteins were ultimately selected for further analysis. Interestingly, four of these proteins corresponded to ASFV enzymes involved in nucleic acid metabolism: G1211R (DNA polymerase β), D1133L (helicase), P1192R (DNA topoisomerase II), and EP424R (putative methyltransferase); while two others corresponded to the p150 and p37 structural proteins, which are precursors to the pp220 polyprotein, encoded by the CP2475L ORF. Additionally, two members of the resulting multigene family 505 were selected: MGF505-1R, which may be involved in IFN-I suppression and is absent in the non-pathogenic OURT88 / 3 and BA71V ASFVs; and MGF505-9R. Finally, the K145R ORF, previously identified as an immunodominant antigen using sera from convalescent pigs, and four additional ORFs of unknown function were selected: B475L, M1249L, H339R, and I226R.

[1184] For each of the 13 selected proteins, encoded in a unique ORF, a computer simulation analysis was performed to identify regions with a high density of epitopes using NetMHCpan 3.0 software as described in Example 1. Finally, a single DNA construct was designed with the selected protein regions linked by an optimal proteasomal cleavage site (AAY) (Velders et al., 2001) and using ubiquitin as a leader sequence to enhance their SLA I processing and presentation. The final plasmid containing the ubiquitin gene encodes a protein of 1,884 amino acids in length and is referred to below as Multi-Epitope-II (ME-II; SEQ ID NO: 856).

[1185] DNA vaccination with plasmids based on multiple ASFV epitopes partially protects against lethal challenge with Georgia 2007 / 1: To test the protective efficacy of selected ASFV candidates, a previously described prime-boost heterologous vaccination regimen was evaluated. Both ME-I and ME-II were administered to pigs. Figure 5 As shown in , three of five pigs (60%) primed with multiple epitopes survived the Georgia 2007 / 01 challenge, whereas only one of five controls (20%) survived, consistent with the expected outcome for the low dose of BA71ΔCD2 used.

[1186] In the multi-epitope-vaccinated group, two of the three surviving animals (pigs 175 and 176) remained afebrile for even two consecutive days, and ASFV-positive samples from these animals' serum and nasal swabs showed low viral titers, confirming the success of the DNA priming with the plasmid. Furthermore, pig 175 remained free of ASF-compatible clinical symptoms throughout the experiment. Animal 176 showed mild lethargy and pronounced dyspnea starting 14 days per week until the end of the experiment, but this was not associated with fever, ASFV-positive serum samples, or nasal shedding. The third survivor (pig 178) showed persistent but mild ASF symptoms and a transient febrile episode starting 5 days per week, associated with prolonged ASFV detection in both serum and nasal swabs. However, by the end of the experiment, this animal had almost recovered, showing no fever and only mild dyspnea and undetectable levels of ASFV in serum and nasal swabs. The rectal temperatures and virus titers of the pigs that had to be sacrificed (177 and 179) from the ME-I + ME-II primed group were not different from those found in the majority of the control animals.

[1187] The course of infection in the control group was consistent with previous results using a low dose of BA71ΔCD2 (Monteagudo et al., 2017). In this group, ASF-compatible clinical symptoms were evident by day 3 after Georgia 2007 / 1 challenge, consistent with prolonged febrile episodes (all animals were febrile for at least five consecutive days) and consistent with viral titers in serum and nasal swabs. Surviving control animals (pig 185) had prolonged fever compared to survivors in the ME-I + ME-II primed group, which also showed delayed onset of ASF symptoms.

[1188] Multi-epitope-based DNA constructs encoding multiple epitopes from ASFV induce ASFV-specific T cells in vivo: As expected, multi-epitope vaccination did not induce any ASFV-specific antibody responses, but it did induce a detectable ASFV-specific IFNγ response, confirming the success of DNA priming with the selected antigens. Following the BA71ΔCD2 boost, all pigs seroconverted and the number of ASFV-specific T cells increased significantly. As previously described, there appeared to be no correlation between the levels of antibodies or specific T cells at the time of challenge and the protection achieved, at least when measured by the techniques employed here.

[1189] To confirm the antigenicity of the multi-epitope DNA construct, IFNγ responses were detected by ELISpot when it was transfected into fibroblasts and these transfected fibroblasts were used as APCs for autologous PBMCs obtained at 21 dpb.

[1190] Recognition of ASFV Antigens: DNA-Primed Animals vs. Unprimed Animals: To determine and rank the immunogenicity of individual ASFV antigens encoded in multi-epitope-based DNA constructs, pCMV-Ub plasmids encoding each of the full-length proteins included in both ME-I and ME-II were transfected into fibroblasts, and these transfected fibroblasts were used as APCs in ELISpot assays. Expression of the full-length ASFV antigens used here was confirmed by immunofluorescence as described in Example 1. To determine the effect that DNA priming can have on the induction and regulation of T cell responses, PBMCs from multi-epitope-primed, non-DNA-primed animals vaccinated with BA71ΔCD2 were used as effector cells in ELISpot assays. While nearly all clones stimulated specific IFN-γ responses, proteins CP312R and D1133L exhibited the most promiscuous properties in both cases. Surprisingly, while 3 of 5 previously unprimed ASFV-infected animals responded to CP312R and D1133L, IFN-γ production was observed in all DNA-primed animals, indicating the effect of DNA priming on the induction of specific T cell responses against these two antigens. An increase in the number of animals responding to A240L was also seen in DNA-primed animals. These results confirm the success of DNA priming using the designed multiple epitopes to characterize two previously undescribed immunodominant ASFV antigens, CP312R and D1133L, with the potential to induce protective T cell responses.

[1191] Conclusion: This study demonstrates the feasibility of a multi-epitope DNA construct in inducing ASFV-specific T cell responses in vivo and increasing survival after lethal challenge with Georgia 2007 / 1 when included in a heterologous prime-boost vaccination regimen using LAV BA71ΔCD2 as a booster. Thus, the protective potential of the epitopes encoded in the DNA plasmids was confirmed, validating antigen selection based on immunopeptidomics studies of ASFV-infected macrophages. The use of allogeneic fibroblasts as APCs in ELISpot assays allowed for the narrowing of potential candidates and the identification of CP312R and D1133L as highly promiscuous antigens.

[1192] All compositions and methods disclosed and advocated herein can be made and performed without undue experimentation according to the present invention. Although the compositions and methods of the present invention have been described with respect to certain aspects, it will be apparent to those skilled in the art that variations can be made to the compositions and methods and steps or sequences of method steps described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the following claims.

[1193] References

[1194] The following references are specifically incorporated herein by reference, to the extent they provide exemplary procedural or other details supplementary to those set forth in the references herein.

[1195] Argilaguet JM et al., Vaccine 2011, 29: 5379-5385

[1196] Argilaguet JM et al., PLoS One 2012, 7: e40942

[1197] Calis J et al., PLoS Comput. Biol. 2013, 9: e1003266

[1198] Chapman DA et al., Emerg Infect Dis. 2011, 17(4): 599-605

[1199] De Villiers EP et al., Virology 2010, 400: 128-136

[1200] Farlow J et al., Virology Journal 2018, 15(1): 190

[1201] Galindo-Cardiel I et al., Virus Res 2013, 173: 180-190

[1202] Gallardo C et al., Vet Microbiol 2013, 162: 32-43

[1203] Jancovich JK et al., J Virol 2018, 92(8):e02219-17

[1204] Jenson JS et al., J Immunol Methods 2000, 242: 33-42

[1205] Lacasta A et al., J Virol 2014, 88: 13322-13332

[1206] Lopera-Madrid J et al.,Vet Immunol Immunopthol 2017,185:20-33

[1207] Monteagudo PL et al., J Virol 2017, 91(21):e01058-17

[1208] Netherton CL et al., Front Immunol. 2019(10):1318

[1209] O'Donnell V et al., J Virol 2015, 89: 6048-6056

[1210] Rodriguez F et al., J Virol 2001, 75:7399-7409

[1211] Rodriguez JM et al., PLoS One 2015;10(11):e0142889

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[1213] Sánchez EG et al., Virus Research 2019, 265: 150-155

[1214] Velders MP et al., J Immunol 2001, 166: 5366-5373

[1215] Uniprot database accession number A0A2X0RVA9

[1216] WO 2015 / 091322

[1217] WO 2017 / 096341

[1218] The scope and spirit of the present invention also include the following technical solutions:

[1219] 1. An immunogenic composition comprising

[1220] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[1221] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[1222] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and

[1223] (d) optionally, one or more pharmaceutically or veterinarily acceptable carriers or excipients, preferably suitable for oral, intradermal, intramuscular or intranasal administration.

[1224] 2. A vaccine or pharmaceutical composition comprising

[1225] (a) one, two or more African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[1226] (b) one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African swine fever virus peptides and / or polypeptides, preferably full-length proteins and / or immunogenic fragments thereof; and / or

[1227] (c) a viral or bacterial vector, preferably a recombinant and / or non-naturally occurring viral or bacterial vector, containing one, two or more African swine fever virus oligonucleotides and / or polynucleotides and / or immunogenic fragments thereof encoding African ...

Claims

1. An immunogenic composition comprising: (a) one or two African swine fever virus polypeptides; or (b) one or two African swine fever virus oligonucleotides encoding African swine fever virus polypeptides; or (c) a viral or bacterial vector containing one or two African swine fever virus oligonucleotides encoding an African swine fever virus polypeptide; and (d) one or more pharmaceutically or veterinarily acceptable carriers or excipients; According to (a), (b) and (c), the African swine fever virus polypeptide is selected from the following group: MGF100-1L shown in SEQ ID NO: 572 and SEQ ID NO:

570.

2. The immunogenic composition of claim 1, wherein according to (b), the one or two African swine fever virus oligonucleotides encoding African swine fever virus polypeptides consist of a nucleic acid sequence selected from the group consisting of: SEQ ID NO:573 and SEQ ID NO:

571.

3. The immunogenic composition of claim 1 , wherein according to (c), the viral or bacterial vector is selected from the group consisting of an African swine fever virus vector, an avian pox virus vector, a canine measles virus vector, a herpes virus vector, a varicella virus vector, Lawsonia spp., and Salmonella spp.

4. The immunogenic composition of claim 1, wherein according to (c), the viral or bacterial vector contains one or two African swine fever virus oligonucleotides encoding one or two African swine fever virus polypeptides, wherein the one or two African swine fever virus oligonucleotides are composed of a nucleic acid sequence selected from the group consisting of: SEQ ID NO:573 and SEQ ID NO:

571.

5. The immunogenic composition of claim 1, wherein the African swine fever virus is selected from the group consisting of BA71, BA71ΔCD2 and Georgia 2007 / 1 virus strains.

6. The immunogenic composition of any one of claims 1-5, wherein the immunogenicity of the immunogenic composition is indicated by an induced IFN-γ response in a porcine IFN-γ ELISpot assay.

7. The immunogenic composition of any one of claims 1 to 5, wherein the immunogenic composition is a vaccine.

8. Use of at least one of (a), (b) and (c) in the preparation of an immunogenic composition for treating and / or preventing at least one African swine fever virus infection, (a) one or two African swine fever virus polypeptides; (b) one or two African swine fever virus oligonucleotides encoding African swine fever virus polypeptides; and (c) a viral or bacterial vector containing one or two African swine fever virus oligonucleotides encoding an African swine fever virus polypeptide; According to (a), (b) and (c), the African swine fever virus polypeptide is selected from the following group: MGF100-1L shown in SEQ ID NO: 572 and SEQ ID NO:

570.

9. Use of at least one of (a), (b) and (c) in the preparation of an immunogenic composition for immunizing a porcine animal against clinical symptoms or disease caused by at least one African swine fever virus in the porcine animal, (a) one or two African swine fever virus polypeptides; (b) one or two African swine fever virus oligonucleotides encoding African swine fever virus polypeptides; and (c) a viral or bacterial vector containing one or two African swine fever virus oligonucleotides encoding an African swine fever virus polypeptide; According to (a), (b) and (c), the African swine fever virus polypeptide is selected from the following group: MGF100-1L shown in SEQ ID NO: 572 and SEQ ID NO: 570; The immunogenic composition does not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal against the at least one African swine fever virus.

10. The use according to claim 9, wherein the clinical symptoms or diseases are selected from the group consisting of African swine fever, death, fever, lethargy, weakness, loss of appetite, recumbency, erythema, cyanotic skin nevus, dysentery, constipation, abdominal pain, respiratory symptoms, vomiting, bleeding, abortion, leukopenia, and thrombocytopenia.

11. Use of at least one of (a), (b) and (c) in the preparation of an immunogenic composition for prime-boost immunization of porcine animals against clinical symptoms or diseases caused by at least one African swine fever virus in said porcine animals, (a) one or two African swine fever virus polypeptides; (b) one or two African swine fever virus oligonucleotides encoding African swine fever virus polypeptides; and (c) a viral or bacterial vector containing one or two African swine fever virus oligonucleotides encoding an African swine fever virus polypeptide; According to (a), (b) and (c), the African swine fever virus polypeptide is selected from the following group: MGF100-1L shown in SEQ ID NO: 572 and SEQ ID NO: 570; wherein the immunogenic composition is used to administer to the porcine animal once or twice for a priming step, and subsequently - after the priming step once or twice, the porcine animal is administered with a live attenuated African swine fever virus for a boosting step; The immunogenic composition and the live attenuated African swine fever virus independently of each other do not cause clinical symptoms of infection, but can induce an immune response that immunizes the porcine animal against at least one African swine fever virus.

12. The use according to claim 11, wherein the clinical symptoms or diseases are selected from the group consisting of African swine fever, death, fever, lethargy, weakness, loss of appetite, recumbency, erythema, cyanotic skin nevus, dysentery, constipation, abdominal pain, respiratory symptoms, vomiting, bleeding, abortion, leukopenia, and thrombocytopenia.

13. The use according to claim 11, wherein the live attenuated African swine fever virus is BA71ΔCD2.

14. The use according to any one of claims 8 to 13, wherein the immunogenic composition is a vaccine.

15. A kit for immunizing porcine animals against diseases associated with at least one African swine fever virus in porcine animals, the kit comprising: (a) a dispenser capable of administering the immunogenic composition to the porcine animal; and (b) the immunogenic composition of any one of claims 1 to 7; and (c) Instruction booklet.

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