OA19132A - A rationally developed African Swine Fever attenuated virus strain protects against challenge with parental virus Georgia 2007 isolate. - Google Patents
A rationally developed African Swine Fever attenuated virus strain protects against challenge with parental virus Georgia 2007 isolate. Download PDFInfo
- Publication number
- OA19132A OA19132A OA1201800542 OA19132A OA 19132 A OA19132 A OA 19132A OA 1201800542 OA1201800542 OA 1201800542 OA 19132 A OA19132 A OA 19132A
- Authority
- OA
- OAPI
- Prior art keywords
- asfv
- a9gl
- auk
- virus
- mutant
- Prior art date
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 87
- 230000002238 attenuated Effects 0.000 title claims abstract description 27
- 208000007407 African Swine Fever Diseases 0.000 title claims abstract description 16
- 230000002633 protecting Effects 0.000 title claims abstract description 10
- 241000701386 African swine fever virus Species 0.000 claims abstract description 48
- 229960005486 vaccines Drugs 0.000 claims abstract description 42
- 241000282898 Sus scrofa Species 0.000 claims abstract description 32
- 150000001413 amino acids Chemical class 0.000 claims description 20
- 229920001184 polypeptide Polymers 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 5
- 238000004166 bioassay Methods 0.000 claims description 4
- 229920002676 Complementary DNA Polymers 0.000 claims 4
- 241000124008 Mammalia Species 0.000 claims 4
- 239000002299 complementary DNA Substances 0.000 claims 4
- 238000002965 ELISA Methods 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 24
- 201000009910 diseases by infectious agent Diseases 0.000 abstract description 13
- 241000282887 Suidae Species 0.000 abstract description 11
- 230000017613 viral reproduction Effects 0.000 abstract description 10
- 230000008506 pathogenesis Effects 0.000 abstract description 9
- 230000001018 virulence Effects 0.000 abstract description 9
- 208000001756 Virus Disease Diseases 0.000 abstract description 2
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 16
- 150000002500 ions Chemical class 0.000 description 13
- 241000701251 African swine fever virus Malawi LIL 20/1 Species 0.000 description 12
- 210000002540 Macrophages Anatomy 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 10
- 238000003780 insertion Methods 0.000 description 9
- 230000002068 genetic Effects 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000006011 modification reaction Methods 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 206010037660 Pyrexia Diseases 0.000 description 6
- 230000000875 corresponding Effects 0.000 description 6
- 210000003702 immature single positive T cell Anatomy 0.000 description 6
- 238000010276 construction Methods 0.000 description 5
- 230000001717 pathogenic Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 210000004027 cells Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000001681 protective Effects 0.000 description 4
- 108010090686 African swine fever virus capsid protein p72 Proteins 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 229920001850 Nucleic acid sequence Polymers 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000002354 daily Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 media Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- 230000002829 reduced Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002194 synthesizing Effects 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 102100001249 ALB Human genes 0.000 description 2
- 101710027066 ALB Proteins 0.000 description 2
- 210000004369 Blood Anatomy 0.000 description 2
- 229920000453 Consensus sequence Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 206010061428 Decreased appetite Diseases 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 102100004985 GUSB Human genes 0.000 description 2
- 206010017577 Gait disturbance Diseases 0.000 description 2
- 108010060309 Glucuronidase Proteins 0.000 description 2
- 235000002912 Salvia officinalis Nutrition 0.000 description 2
- 206010040829 Skin discolouration Diseases 0.000 description 2
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L Thiomersal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 230000000240 adjuvant Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229940050528 albumin Drugs 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000000712 assembly Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 201000008286 diarrhea Diseases 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 230000001894 hemadsorption Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 230000000670 limiting Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 244000052769 pathogens Species 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 230000002335 preservative Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 235000002020 sage Nutrition 0.000 description 2
- 239000001296 salvia officinalis l. Substances 0.000 description 2
- 230000037370 skin discoloration Effects 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000001052 transient Effects 0.000 description 2
- 210000003165 Abomasum Anatomy 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 230000036849 Clc Effects 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 210000000805 Cytoplasm Anatomy 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102200033352 FOXC1 A85P Human genes 0.000 description 1
- 241000272184 Falconiformes Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 206010018987 Haemorrhage Diseases 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N Heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960002897 Heparin Drugs 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 229940047124 Interferons Drugs 0.000 description 1
- 241000701377 Iridoviridae Species 0.000 description 1
- 102220379521 LIN9 E224Q Human genes 0.000 description 1
- 210000000265 Leukocytes Anatomy 0.000 description 1
- 210000002433 Leukocytes, Mononuclear Anatomy 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 210000001132 Macrophages, Alveolar Anatomy 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 210000001616 Monocytes Anatomy 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 241000701253 Phycodnaviridae Species 0.000 description 1
- 241000700625 Poxviridae Species 0.000 description 1
- 239000007759 RPMI Media 1640 Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229940033663 Thimerosal Drugs 0.000 description 1
- 210000003501 Vero Cells Anatomy 0.000 description 1
- 206010047461 Viral infection Diseases 0.000 description 1
- 210000002845 Virion Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000001154 acute Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- -1 dried milk sérum Chemical class 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005188 flotation Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulators Drugs 0.000 description 1
- 230000002458 infectious Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated Effects 0.000 description 1
- 238000002941 microtiter virus yield reduction assay Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000036961 partial Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000023 polynucleotide Polymers 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 238000001303 quality assessment method Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
Abstract
African swine fever virus (ASFV) is the etiological agent of a contagious, often letha! viral disease of domestic pigs. The control of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Expérimental vaccines have been derived from naturally occurring, cell cultureadapted, or genetically modified live attenuated ASFVs; however, these vaccines are only successful when protecting against homologous viruses. We have constructed a recombinant A9GL/AUK virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate by deieting the specific virulence-associated 9GL (B119L) and the UK (DP96R) genes. in vivo, ASFV-G A9GL/AUK administered intramuscularly to swine even at relatively high doses (106 HAD50) does not induce disease. Importantly, animais infected with 104 or (106 HAD50) are solidly protected against the présentation of clinical disease when challenged at 28 days post infection with the virulent parentai strain Georgia 2007.
Description
The United States of America às represented by the Secretary of Agriculture and The University of Connecticut
A RationaOy Developed African Swine Fever Attenuated Virus Strain Protects Agamst Chahenge With Parental Virus Georgîa 2007 isoîate
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention relates to the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated candidate strain vaccine for the highly virulent Georgia 2007 isolate ASFV-G. The vaccine comprises the ASFV-G A9GLAUK modified virus, a recombinant ASFV-G modified by deleting a large portion of the 9GL (B119L) gene and the UK (DP96R) gene.
Description of the ·Relevant Art
[0002] African Swine Fever (ASF) is a contagious vira' disease of swine. The causative agent, ASF virus (ASFV), is a large enveloped virus containing a doubîe-stranded DNA genome of approximately 190 kilobase pairs. ASFV shares aspects of genome structure and réplication strategy with other large double-stranded DNA viruses, including the Poxviridae, Iridoviridae and Phycodnaviridae (Costard et ai 2009. Phil. Trans. Royal Soc. B 364:2683=2696). ASFV infections in domestic pigs are often fatal and are characterized by fever, hemorrhages, ataxia and severe dépréssion. However, the course of infection varies, ranging from highly léthal to sub-clinical, depending on host characteristics and the particular virus strain (Tulman et al. 2009. Cuit. Top. Microbiol. Immunol. 328:43-87),
[0003] Currently, the disease is endemic in more than twenty sub=Saharan African countries. in Europe, ASF is stiii endemic on the island of Sardinia (italy) and new
19131 outbreaks hâve been declared in the Caucasus région since 2007, affecting Georgia, Armenia, Azerbaijan and Russia. Isolated outbreaks hâve been recently reported in Ukraine, Bélarus, Lithuania, Latvia and Roland, posing the risk of further dissémination into neighbouring countries. The épidémie virus, ASFV Georgia 2007/1, is a highly virulent isolate belonging to the génotype II (Chapman et al. 2011. Emerging Infect. Dis. 17:599-605).
[0004] Currently, there is no vaccine available for ASF and disease outbreaks are controlled by animal quarantine and slaughter. Attempts to vaccinate animais using infected cell extracts, supernatants of infected pig peripheral blood leukocytes, purified and inactivated virions, infected glutaraldehyde-fixed macrophages, or detergent-treated infected alveolar macrophages failed to induce protective immunity (Coggins, L. 1974. Prog. Med. Virol. 18:48-63; Forman étal. 1982. Arch. Virol. 74:91-100; Kihm étal. 1987. In: African Swine Fever, Becker, Y. (ed), Martinus Nijhoff, Boston, pp 127-144; Mebus, C. A. 1988. Adv. Virus Res. 35:251—269). Homologous protective immunity does develop in pigs surviving viral infection. Pigs surviving acute infection with moderately virulent or attenuated variants of ASFV develop long-term résistance to homologous, but rarely to heterologous, virus challenge (Hamdy and Dardiri. 1984. Am. J. Vet. Res. 45:711-714; Ruiz-Gonzalvo et al. 1981. In: FAO/CEC Expert Consultation in ASF Research, Wilkinson, P. J. (ed), Rome, pp 206-216). Pigs immunized with live attenuated ASF viruses containing engineered délétions of spécifie ASFV virulenceassociated genes were protected when challenged with homologous parental virus. Specifically, individual délétion of UK (DP69R), 23-NL (DP71L), TK (A240L) or 9GL (B119L) genes from the genomes of pathogenic ASF viruses (Malawi Lil-20/1, Pretoriuskop/96/4, E70 and Georgia 2007) markedly attenuated the virus in swine and the animais immunized with these attenuated viruses were protected against challenge with homologous virus (Moore étal. 1998. J. Virol. 72:10310-10315; Lewis étal. 2000. J. Virol. 74:1275-1285; Zsak étal. 1996. J. Virol. 70:8865-8871; Zsak étal. 1998. J.
19131
Virol. 72:1028-1035). These observations constitute the only experimental evidence describing the rational development of an effective live attenuated virus against ASFV.
[0005] In particular, délétion of 9GL (B119L) in highly virulent ASFV isolâtes Malawi Lil20/1, Pretoriuskop/96/4, and Georgia2007 (Lewis étal., supra-, Neilan étal. 2004. Virol. 319:337-342; O Donnell et al. 2015. J. Virol. 89: 8556-8566) resulted in complété atténuation of these viruses in swine. Administration of Malawi Lil-20/1A9GL or Pretoriuskop/96/4 A9GL or the E70 AUK mutants to pigs via IM injection at a relatively high virus dose did not induce clinical signs, with ail animais surviving the infection. Furthermore, IM inoculation of pigs with these viruses induced protection against challenge with virulent parental viruses (Zsak et al. 1998, supra-, Lewis et al., supra; O’Donnell et al., supra). These observations constitute the only experimental evidence describing the rational development of an effective live attenuated virus against ASFV.
[0006] Since there are not ASFV vaccines currently available, the development of any experimental vaccine that may induce any type of protection against the léthal présentation of the disease is of great interest.
SUMMARY OF THE INVENTION
[0007] We hâve developed the novel recombinant mutant ASFV-G A9GL/AUK virus, a modification of the ASFV-G (African Swine Fever Virus- Georgia 2007 isolate).
[0008] In accordance with this discovery, it is an object of the invention to provide the novel mutant ASFV-G A9GL/AUK virus, resulting from the délétion of a large portion of both the 9GL (B119L) gene and the UK (DP96R) gene of the parental ASFV-G. The nucléotide sequence of ASFV-G A9GL/AUK (SEQ ID NO: 3) differs from the nucléotide sequence encoding the wild-type ASFV-G (SEQ ID NO: 1). The ASFV-G (wild-type) 9GL- encoded protein of 119 amino acids (SEQ ID NO: 2) and the ASFV-G (wild-type) UK - encoded protein of 95 amino acids (SEQ ID NO:25) differfrom the mutant 9GL and
19131
UK proteins encoded by the mutant nucléotide sequence of ASFV-G A9GL/AUK (SEQ ID NO: 3). A mutant 9GL polypeptide of 61 amino acids (SEQ ID NO: 4) results from the délétion of amino acid # 11 through amino acid #68 of the wild-type 9GL polypeptide (SEQ ID NO: 2), and a mutant UK polypeptide of 10 amino acids (SEQ ID NO: 26) results from the délétion of amino acid #1 through amino acid #85 of the wild-type UK polypeptide (SEQ ID NO:25).
[0009] An added object of the invention is to provide immunogenic compositions comprising a viable ASFV-G A9GL/AUK virus.
[0010] An additional object of the invention is to provide a rationally designed live attenuated ASFV-G A9GL/AUK vaccine effective to protect an animal from clinical ASF disease when challenged with pathogenic ASFV-G.
[0011] A further object of the invention is to provide a genetic marker vaccine which can potentially distinguish between vaccinated animais and animais infected with ASFV-G.
[0012] Another object of the invention is to provide a method for protecting an animal against ASFV-G by administering an effective amount of rationally designed live attenuated ASFV-G A9GL/AUK vaccine.
[0013] An additional object of the invention is to provide a method for distinguishing animais infected with ASFV-G from animais vaccinated with said rationally designed live attenuated ASFV-G A9GL/AUK vaccine, comprising a genetic DIVA strategy for differentiating vaccinated animais from wild-type infected animais.
[0014] Other objects and advantages of this invention will become readily apparent from the ensuing description.
19131
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figures 1A and 1B show the sequence alignment of ASFV-G 9GL (B119L) geneencoded polypeptides (Fig. 1A), and the ASFV-G UK (DP96R) gene-encoded polypeptides (Fig. 1B). Isolâtes of various temporal and géographie origins, including those from obtained from ticks and pig sources, were compared. The partial délétion introduced into ASFV-G that yielded ASFV-G A9GL/AUK virus is shown between brackets.
DETAILED DESCRIPTION OF THE INVENTION
[0016] We hâve developed an attenuated virus that can be used as a vaccine candidate through the approach of targeting both ASFV-G 9GL (B119L) and ASFV-G UK(DP96R) genes for genetic modifications. Here we report the construction of a recombinant A9GL/AUK virus of the highly virulent ASFV Georgia 2007 isolate (ASFV-G). ASFV-G A9GL/AUK administered intramuscularly (IM) to swine at relatively high doses (104 or 106 HAD50) does not induce disease. Animais infected with 104 or 106 HAD50 are protected against the présentation of clinical disease when challenged at 28 days post infection with the virulent parental strain Georgia 2007.
[0017] Although independent délétion of four different genes from ASFV has been shown to attenuate virulent viruses and although independent délétions of the NL (DP71L) (Zsak et al. 1996, supra) or the UK (DP69R) (Zsak et al. 1998, supra) genes from ASFV E75, délétion of the TK (A240L) gene (Moore et al., supra) from ASFV adapted to Vero cells, Malawi Lil-20/1 and Haiti, and délétion of the 9GL (B119L) gene also from Malawi Lil-20/1 (Lewis et al., supra) and Pretoriuskop/96/4 (Neilan et al., supra) isolâtes rendered recombinant délétion mutant viruses with significantly reduced virulence in swine, in ail these cases, animais inoculated with each of these genetically modified viruses survived the infection and became protected against ASFV when
19131 challenged with the corresponding virulent parental virus, i.e., homologous challenge (Lewis étal., supra; Moore étal., supra; Neilan étal., supra; Zsak étal. 1996, supra; Zsak et al. 1998, supra). Those findings suggest that development of attenuated ASFV recombinant viruses by genetic manipulations of target genes is an effective approach for vaccine development.
[0018] However, their level of effectiveness in other ASFV isolâtes is not predictable. For example, the NL (DP71L) gene product exits in two different forms, a long (184 amino acids as in 23-NL) or a short form (70 to 72 amino acids) depending on the ASFV isolate (Zsak et al. 1996, supra). Although délétion of this gene in ASFV E70 isolate (short form) rendered an attenuated virus, the délétion of the NL (DP71 L) gene from ASFV Malawi Lil-20/1 (long form) or Pretoriuskop/96/4 (short form) did not resuit in atténuation of the virus (Afonso étal. 1998. J. Gen. Virol. 79 (Pt. 10):2543-2547). Délétion of the TK (A240L) gene, a highly conserved gene among ail ASFV isolâtes involved in DNA synthesis, has been introduced into pathogenic Vero cell-adapted Malawi Lil-20/1 and Haiti H811 viruses. The Malawi Lil-20/1 mutant virus was less virulent in vivo than the revertant virus (wild-type-like virus), but it was not completely attenuated (Moore et al., supra). The UK (DP69R) gene is located in the right variable région of certain ASFV isolâtes. Délétion of this gene from ASFV E70 isolâtes rendered a virus exhibiting reduced virulence (Zsak et al. 1998, supra). Although the UK (DP69R) gene is conserved, it is not présent in every ASFV isolate (e.g. Malawi Lil20/1), limiting its use as a candidate target gene for producing attenuated viruses.
[0019] The 9GL (B119L) gene is highly conserved among ASFV isolated and sequenced so far, including those from both tick and pig sources. The fact that délétion of the gene from virulent Malawi Lil-20/1 (Lewis et al., supra) and Pretoriuskop/96/4 (Neilan et al., supra) effectively reduced virulence in swine and induced protection made 9GL (B119L) a strong candidate target gene for modification and production of attenuated virus that can confer effective protection against ASFV. Indeed, we found
19131 that the délétion of 9GL (B119L) from the ASFV-G isolate did not hâve the saine effect in terms of atténuation and protection as reported for Malawi Lil-20/1 and Pretoriuskop/96/4. Only when ASFV-G A9GL was administrated at a low dose to swine was it possible to observe a decrease in virus virulence (O’Donnell et al., supra). \Ne had also shown that a sub-lethal dose of ASFV-G A9GL was able to induce effective protection against the présentation of clinical disease after the challenge with homologous parental virus. The NL proteins encoded by E70 (short form) and Malawi Lil-20/1 (long form) differ significantly and that may explain the phenotypic différences observed in swine inoculated with the respective délétion mutant viruses. However, protein identity matrixes indicate that the 9GL protein is highly similar among ASFV isolâtes where ASFV-G, Malawi Lil-20/1, and Pretoriuskop/96/4 share over 93% amino acid identity, making it unlikely that ASFV atténuation relies solely on protein divergence. Since the well observed phenotypes are most likely mediated by the effect of multiple genes (Lewis et al., supra; Moore et al., supra; Neilan et al., supra; Zsak et al. 1996, supra; Zsak et al. 1998, supra), the evidence accumulated so far makes it difficult to speculate what is indeed the spectrum of genes mediating virulence in the ASFV Georgia 2007 isolate.
[0020] In summary, here we présent evidence that délétion of both the 9GL (B119L) gene and the UK (DP69R) gene results in the attenuated recombinant ASFV-G A9GL/AUK virus. Intramuscular administration of ASFV-G A9GL/AUK to swine at relatively high doses (104 or 106 HAD50) does not induce disease. Animais infected with 104 or 106 HAD50 are protected against the présentation of clinical disease when challenged at 28 days post infection with the virulent parental strain Georgia 2007.
[0021] A vaccine is defined herein as a biological agent which is capable of providing a protective response in an animal to which the vaccine has been delivered and is incapable of causing severe disease. Administration of the vaccine results in immunity from a disease; the vaccine stimulâtes antibody production or cellular immunity against
19131 the pathogen causing the disease. Immunity is defined herein as the induction of a significant higher level of protection in a population of swine against mortality and clinical symptoms after vaccination compared to an unvaccinated group. In particular, the vaccine according to the invention protects a large proportion of vaccinated animais against the occurrence of clinical symptoms of the disease and mortality. The vaccine of the invention herein is a genetically engineered mutant virus vaccine. A genetic marker vaccine is defined as a vaccine that, in conjunction with a diagnostic test, enables genetic différentiation of vaccinated animais from infected animais. A délétion mutation can be used to differentiate infected from vaccinated animais. A mutation is understood to be a change in the genetic information of a wild-type or unmodified 9GL (B119L) and UK (DP96R) genes of a parent ASFV-G strain which is able to express native 9GL and UK proteins. Thus, the 9GL and UK polypeptides expressed by the ASFV-G A9GL/AUK mutant virus is changed: the 9GL protein from ASFV-G A9GL/AUK has fewer amino acids than both the wild-type 9GL and the wild-type UK, as amino acids #11 through #68 are deleted in the 9GL polypeptide of ASFV-G A9GL and amino acids #1 to #85 are deleted in the UK polypeptide. The ASFV-G A9GL/AUK recombinant ASFV-G mutant comprises nucléotides encoding mutations in the ASFV-G 9GL and UK polypeptides. The mutation comprises a délétion of 58 amino acids of the 9GL protein and a délétion of 85 amino acids of the UK protein. The recombinant ASFV-G mutant ASFV-G A9GL/AUK is a live attenuated ASFV-G vaccine when used at IM inoculation doses of 104 HAD50to 106 HAD50.
[0022] A vaccine against ASFV-G is provided that comprises a ASFV-G A9GL/AUK mutant as defined above in a live form, and a pharmaceutically acceptable carrier or diluent. The vaccine according to the invention containing the live virus can be prepared and marketed in the form of a suspension or in a lyophilized form and additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions. Carriers include stabilizers, preservatives and buffers. Suitable stabilizers are, for example SPGA (sucrose, phosphate, glutamate, and human. albumin),
19131 carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk sérum, albumin or casein) or dégradation products thereof. Suitable buffers are for example alkali métal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
[0023] If desired, the live vaccines according to the invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are well known in the art. Furthermore, nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic applications, in particular immunomodulators such as lymphokines, interferons or cytokines, may be incorporated into the vaccine.
[0024] A vaccine according to the invention can be prepared by conventional methods such as those commonly used for the commercially available live attenuated ASFV vaccines. Briefly, a susceptible substrate is inoculated with the ASFV-G A9GL/AUK mutant and propagated until the virus has replicated to a desired titer after which ASFVG A9GL/AUK -containing material is harvested. Subsequently, the harvested material is formulated into a pharmaceutical préparation with immunizing properties.
[0025] Every substrate which is able to support the réplication of ASFV-G A9GL/AUK viruses can be used in the présent invention, including primary cultures of swine peripheral blood macrophages.
[0026] The vaccine may be administered by intramuscular, subcutaneous or intranasal inoculation or injection in an amount which is effective to protect the animal against challenge by a virulent strain of ASFV-G. This amount may vary according to the animal being inoculated, taking into considération the size and weight of the animal. The vaccine according to the invention comprises an effective dosage of the ASFV-G A9GL/AUK mutant as the active component, Le. an amount of immunizing ASFV-G
19131
A9GL/AUK material that will induce immunity in the vaccinated animais, swine, against challenge by a virulent ASFV-G. Immunity is defined herein as the induction of a significant higher level of protection in a population of swine against mortality and clinical symptoms after vaccination compared to an unvaccinated group. In particular, the vaccine according to the invention prevents a large proportion of vaccinated animais against the occurrence of clinical symptoms of the disease and mortality. Typically, the live vaccine can be administered in a dose of 104 HAD50to 106 HAD50. Effective amounts may be experimentally determined as necessary by those of skill in the art by following the guidance provided, for example, by Example 6.
[0027] In addition to the ASFV-G A9GL/AUK mutant, the invention can also include combination vaccines comprising a vaccine strain capable of inducing protection against another porcine pathogen.
[0028] The ASFV-G A9GL/AUK vaccine described above, in conjunction with a diagnostic method, has the potential of distinguishing between animais that are vaccinated with it and animais that are infected with naturally occurring ASFV-G strains or vaccinated with conventional ASFV-G vaccines.
[0029] The présent invention also provides an invaluable tool to monitor ASFV-G control measures that may lead to éradication of ASFV-G if applied in large scale stamping out programs. This tool concerns a method for determining ASFV-G infection in swine comprising the step of examining a sample of the animal for the presence of nucléotides encoding the wild-type ASFV-G 9GL and UK proteins versus the polynucleotide encoding the shorter ASFV-G A9GL and AUK polypeptides due to délétions in the 9GL (B119L) gene and the UK (DP96R) genes of ASFV-G A9GL/AUK. The sample of the animal used in this method may be any sample in which ASFV-G
19131 versus ASFV-G A9GL/AUK genetic différences allowing for differentiating of natural infection versus vaccination can be detected by genetic DIVA.
EXAMPLES
[0030] Having now generally described this invention, the same will be better understood by reference to certain spécifie examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the daims.
EXAMPLE 1
Cell Cultures and Viruses
[0031] Primary swine macrophage cell cultures were prepared from defibrinated swine blood as previously described by Zsak et al. (1996, supra). Briefly, heparin-treated swine blood was incubated at 37°C for 1 hour to allow sédimentation ofthe érythrocyte fraction. Mononuclear leukocytes were separated by flotation over a Ficoll-Paque (Pharmacia, Piscataway, N.J.) density gradient (spécifie gravity, 1.079). The monocyte/macrophage cell fraction was cultured in plastic Primaria (Falcon; Becton Dickinson Labware, Franklin Lakes, N.J.) tissue culture flasks containing macrophage media, composed of RPMI 1640 Medium (Life Technologies, Grand Island, NY) with
19131
30% L929 supernatant and 20% fêtai bovine sérum (HI-FBS, Thermo Scientific, Waltham, MA) for 48 hours at 37°C under 5% CO2. Adhèrent cells were detached from the plastic by using 10 mM EDTA in phosphate buffered saline (PBS) and were then reseeded into Primaria T25, 6- or 96-well dishes at a density of 5x106 cells per ml for use in assays 24 hours later.
[0032] Virus titration was performed on primary swine macrophage cell cultures in 96well plates. Virus dilutions and cultures were performed using macrophage medium. Presence of virus was assessed by hemadsorption (HA) and virus titers were calculated by the Reed and Muench method (1938. Amer. J. Hygiene 27:493-497).
[0033] ASFV Georgia (ASFV-G) was a field isolate kindly provided by Dr. Nino Vepkhvadze, from the Laboratory of the Ministry of Agriculture (LMA) in Tbilisi, Republic of Georgia.
EXAMPLE 2
Construction of the recombinant ASFV-G A9GL/AUK
[0034] Recombinant ASFVs were generated by sequential homologous recombination between the parental ASFV genome and recombination transfer vectors in infection and transfection procedures using swine macrophage cell cultures (Neilan étal., supra-, Zsak étal. 1996, supra). First, recombinant transfer vector (p72GUSA9GL) containing flanking genomic régions including portions of 9GL mapping to the left (1.2 kbp) and right (1.15 kbp) of the gene and a reporter gene cassette containing the β-glucuronidase (GUS) gene with the ASFV p72 late gene promoter, p72GUS was used. This construction created a 173-nucleotide délétion in the 9GL ORF (amino acid residues 11
19131 to 68) (see Fig. 1). Recombinant transfer vector p72GUSA9GL was obtained by DNA synthesis (GenScript, Piscataway, NJ, USA). Macrophage cell cultures were infected with ASFV-G and transfected with p72GUSA9GL. Recombinant viruses representing independent primary plaques were purified to homogeneity by successive rounds of plaque assay purification. The recombinant virus was obtained after 11 successive plaque purification events on monolayers of primary swine macrophage cell cultures. The produced intermediate recombinant virus, ASFV-G A9GL, was then used as parental virus in infectious/transfection procedures using a recombinant transfer vector that would produce délétion of the UK gene from the virus genome. Recombinant transfer vector (p72mCherryAUK) containing flanking genomic régions of UK mapping to the left (1.156 kbp) and right (1.190 kbp) of the gene and a reporter gene cassette containing the mCherry gene with the ASFV p72 late gene promoter, p72mCherry was used. Recombinant transfer vector p72mCheryAUK was obtained by DNA synthesis (GenScript, Piscataway, NJ, USA). This construction created a 255-nucleotide délétion in the UK ORF (amino acid residues 1 to 85) (see Fig. 1). The second recombination event replaced the UK gene by the cassette containing the fluorescent gene mCherry under the ASFV p72 promoter. Recombinant virus was selected after 10 rounds of limiting dilution purification based in the fluorescent activity. The virus population obtained from the last round of purification was amplified in primary swine macrophage cell cultures to obtain a virus stock.
EXAMPLE 3
Full Genome Sequence Analysis: ASFV-G A9GL/AUK Relative to Parental ASFV-G [0035] To evaluate the accuracy of the genetic modification and the integrity of the genome of the recombinant virus, full genome sequences of ASFV-G A9GL/AUK and parental ASFV-G were obtained using Next Génération Sequencing (NGS) and
19131 comparée! (Table 1). First, a full-length genome comparison between parental ASFV-G and ASFV Georgia 2007/1 (Chapman et al., supra) was performed. ASFV DNA was obtained from the cytoplasm of infected cells using the Trizol method (Life Technologies, Grand Island, NY, USA). DNA concentration was determined using the Qubit® dsDNA HS assay kit (Life Technologies) and read on a Qubit® 2 Flourometer (Life Technologies). One microgram of virus DNA was enzymatically fragmented to obtain blunt end fragments in a length range of 200-300 bp using the Ion Shear™ Plus reagent kit (Life Technologies) and incubated at 37°C in a Peltier Thermal Cycler DNA Engine Tetrad 2. After shearing, the fragmented DNA library was loaded onto a DNA chip (Agilent, Santa Clara, CA, USA) and analyzed using a 2100 Bioanalyzer (Agilent) to assess DNA size distribution and size range. Fragmented DNA was ligated to Ioncompatible adapters and library barcodes, followed by nick-repair to complété the linkage between adapters and DNA inserts using the Ion Plus Fragment Library kit (Life Technologies). The adapter-ligated library was size-selected for optimum length on 2% Agarose Gel Cassettes (Sage Science, Beverly, MA, USA) using the Pippin Prep™ instrument (Sage Science). Library concentration was normalized using the Ion Library Equalizer™ Kit (Life Technologies). Next, the DNA library was clonally amplified onto Ion Sphere™ Particles (IPS) generating template-positive ISPs using the Ion PGM™ Template OneTouch™ 2 200 Kit (Life Technologies) with the Ion OneTouch™ 2 Instrument (Life Technologies). Before proceeding to enrichment, quality assessment of non-enriched template-positive ISPs was performed using the Ion Sphere™ Quality Control assay kit (Life Technologies) and a Qubit® 2 Flourometer instrument. The template-positive ISPs were then enriched using the Ion PGM™ Template OneTouch™ 2 200 Kit (Life Technologies) and Ion OneTouch™ ES instrument (Life Technologies) to eliminate template-negative ISPs and to dénaturé DNA on template-positive ISPs. Using the Ion PGM™ 200 Sequencing v2 Kit (Life Technologies), enriched template ISPs were prepared for sequencing and loaded onto either Ion 314™ or Ion 316™ Chip v2 (Life Technologies) and run on the Ion PGM™ Sequencer (Life Technologies). Obtained sequences were then trimmed using Galaxy (Retrieved from the Internet:
19131 usegalaxy.org/) NGS QC and Manipulation tools. Sequences were aligned and analyzed using Sequencher 5.2.2 (Genecodes) and CLC Genomics Workbench (CLCBio) software.
[0036] The following différences were observed between these two viruses (nucléotide positions are provided based on ASFV Georgia 2007/1, GenBank accession FR682468): (i) two nucléotide insertions, T at position 433 and A at position 441 in a non-coding segment of the genome; (ii) two nucléotide délétions, T at position 1602 and T at position 1603 in the MGF 360-1L gene ORF resulting in a frameshift; (iii) a nucléotide délétion, T at position 1620 in the MGF 360-1L gene ORF resulting in a frameshift; (iv) a nucléotide mutation, A to G at position 97391 resulting in a silent mutation in ORF B438L; (v) a nucléotide mutation, C to G at position 166192 resulting in a residue substitution (Ala to Pro) at residue position 85 in ORF E199L; and (vi) a nucléotide insertion, T at position 183303, a non-coding segment of the genome (Table 1). Second, a full-length genome comparison between ASFV A9GL/AUK and parental ASFV-G was performed. The DNA sequence assemblies of ASFV A9GL/AUK and ASFV-G revealed a délétion of 173 nucléotides in 9GL gene corresponding with the introduced modification. The consensus sequence of the ASFV A9GL/AUK genome showed an insertion of 2324 nucléotides in 9GL gene corresponding to the p72-pGUS cassette sequence introduced to generate a 173 nucléotide délétion in the targeted gene. In addition, the DNA sequence assemblies of ASFV-G A9GL/AUK and ASFV-G revealed a délétion of 255 nucléotides in UK gene corresponding with the introduced modification. The consensus sequence of the ASFV-G A9GL/AUK genome showed an insertion of 937 nucléotides in UK gene corresponding to the p72-mCherry cassette sequence introduced to generate a 255 nucléotide délétion in the targeted gene. Besides the insertion of the cassette, only one additional différence was observed between ASFV-G A9GL/AUK and ASFV-G genomes, a G to C nucléotide mutation at position 36,465 resulting in a residue substitution (Glu to Gin) at residue position 224 in ORF MGF 505-4R. In summary, ASFV-G A9GL/AUK virus did not accumulate any
19131 significant mutations during the process of homologous recombination and plaque purification (Table 1).
Table 1. Summary of différences between the full-length genome sequence of ASFV-G A9GL/AUK and the parental ASFV-G compared with ASFV Georgia07/1*
| Virus | |||
| NPN* | Type of Modification | ASFV-G | ASFV-G A9GL/AUK |
| 433 | T insertion | + | + |
| 411 | A insertion | + | + |
| 1602 | MGF 360-1L TT délétion FS® | + | + |
| 1620 | MGF 360-1L T insertion FS | + | + |
| 36465 | MGF 505-4R G to C Glu224Gln | - | + |
| 97391 | B438L A to G SM# | + | + |
| 166192 | E199L C to G Ala85Pro | + | + |
| 183303 | T insertion in a NCR+ | + | + |
Nucléotide Position Number (based on the sequence of ASFV Georgia 2007/1 isolate published by Chapman et al. 2011) @ Nucléotide modification causes frameshift in the corresponding ORF # Nucléotide modification causes silent mutation + Non-Coding Région
EXAMPLE 4
Assessment of ASFV-G A9GL/AUK Virulence in Swine
19131
[0037] Animal experiments were performed under biosafety level 3 conditions in the animal facilities at PIADC following a protocol approved by the Institutional Animal Care and Use Committee.
[0038] ASFV-G A9GL/AUK was assessed for its virulence phenotype relative to the virulent parental ASFV-G virus using 80-90 pound commercial breed swine. Five pigs were inoculated intramuscularly (IM) either with 104 or 106 HAD50 of either ASFV-G A9GL/AUK or with 104 HAD50 of ASFV-G virus. Clinical signs (anorexia, dépréssion, fever, purple skin discoloration, staggering gait, diarrhea and cough) and changes in body température were recorded daily throughout the experiment. In protection experiments animais were IM inoculated with 104 HAD50 or 106 HAD50 and 28 days later IM challenged with 103 HAD50 of parental virulent ASFV Georgia2007 strain. Presence of clinical signs associated with the disease was performed as described earlier.
[0039] Ail 80-90 pounds pigs inoculated via IM with 104HAD50 of ASFV-G exhibited increased body température (>104 °F) by 3 to 4 days post-infection. Pigs presented clinical signs associated with the disease including anorexia, dépréssion, purple skin discoloration, staggering gait and diarrhea (Table 2). Signs of the disease aggravated progressively over time and animais either died or were euthanized in extremis by days 7 or 9 post-infection. Conversely, animais inoculated via IM with either 104or 106 HAD50 of ASFV-G A9GL/AUK did not présent any signs of clinical disease during the entire observation period (21 days). Therefore, délétion of 9GL and UK genes produced a complété atténuation of the parental virulent ASFV-G. Délétion of UK gene enlarges atténuation of the ASFV-G A9GL to the extent that 106 HAD50 of ASFV-G A9GL/AUK is completely attenuated while animais inoculated with 104 HAD50 of ASFV-G A9GL presented a variable level of disease (O’Donnell et al., supra).
19131
Table 2. Swine survival and fever response following infection with ASFV-G A9GL/AUK and parental ASFV-G viruses.
| Virus | No. of Survivors /Total | Mean Time to death (Days ± SD) | Fever | ||
| No. of Days to onset (Days ± SD) | Duration No. of Days (Days ± SD) | Maximum Daily Temp (°F ± SD) | |||
| ASFV-G 102HAD50 ASFV-G A9GL/AUK | 0/5 | 7.6 (0.55) | 3.6 (0.55) | 4 (0.5) | 105.6 (0.7) |
| 104HAD50 ASFV-G A9GL/AUK | 5/5 | - | - | - | 103.2 (0.32) |
| 106HAD50 | 5/5 | - | - | - | 103.4 (0.59) |
EXAMPLE 6
Protective Efficacy of ASFV-G A9GL/AUK Against Challenge with Parental ASFV-G
[0040] Since pigs inoculated via IM with 104 HAD50-106 HAD50 of ASFV-G A9GL/AUK survived the infection without signs ofthe disease, groups of animais (n=5) infected with either 104 or 106 HADsoof ASFV-G A9GL/AUK were challenged via IM with 1O3HAD5o of parental ASFV-G at day 28 post-inoculation (homologous challenge). Five naïve animais that were challenged using same route and dose served as non-inoculated/ challenged control group. The five ASFV-G A9GL/AUK-inoculated and challenged animais remained completely asymptomatic during ail the observational period (21 days) with the exception of two animais immunized with 106 HAD50 of ASFV-G A9GL/AUK showing a slight and transient rise in body température (Table 3). Ail the animais in the mock inoculated/challenged control group developed disease with a clinical course similar to that observed in animais inoculated with 104HAD50 of ASFV-G
19131 (see above). Therefore, ASFV-G A9GL/AUK is able to induce protection against the présentation of clinical disease when challenged with the highly virulent parental virus.
Table 3. Swine survival and fever response in ASFV-G A9GL/AUK- infected animais challenged with parental ASFV-G viruses.
| Virus | No. of Survivors / Total | Mean Time to death (Days ± SD) | Fever | ||
| No. of Days to onset (Days ± SD) | Duration No. of Days (Days ± SD) | Maximum Daily Temp (F° ± SD) | |||
| Mock | 0/5 | 7.6 (0.55) | 3.6 (0.55) | 5 (0.50) | 105.6(0.7) |
| ASFV-G A9GL/AUK 104 HAD50 | 5/5 | - | - | 102.7 (0.63) | |
| ASFV-G A9GL/AUK 106 had50 | 5/5 | - | 10.5 (9.45)* | 4 (2.84)* | 103.6 (1.21) |
Data are based in 2 out 5 animais presenting transient rise of body température.
#The animais IM infected with 104or 106 HAD50 of ASFV-G A9GL/AUK were IM challenged 28 days laterwith 103 HAD50 of ASFV-G virus.
[0041] In summary, here we présent evidence that délétion of the 9GL and L/Kgenes drastically alter virulence of ASFV-G producing a completely attenuated virus named ASFV-G A9GL/AUK. Animais immunized with ASFV-G A9GL/AUK were protected against challenge with the virulent parental ASFV-G.
[0042] AH publications and patents mentioned in this spécification are herein incorporated by reference to the same extent as if each individual publication or patent
19131 was specifically and individually indicated to be incorporated by reference.
[0043] The foregoing description and certain représentative embodiments and details of the invention hâve been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the précisé forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.
Claims (8)
- ClaimsWe claim:1. A recombinant ASFV-G (African Swine Fever Virus- Georgia 2007 isolate) mutant, the mutant ASFV-G A9GL/AUK virus, comprising cDNA encoding mutant ASFV-G A9GL/AUK polypeptides wherein the mutant cDNA comprises two délétions, a délétion of 173 nucléotides resulting in a mutant 9GL protein comprising 58 fewer amino acids than the non-mutated, wild-type 9GL protein of ASFV-G, amino acids #11 to #68 being deleted, and a second délétion of 255 nucléotides resulting in a mutant UK protein comprising 85 fewer amino acids than the non-mutation, wild-type UK protein of ASFVG, amino acids # 1 to #85 being deleted.
- 2. The mutant cDNA of Claim 1 wherein said cDNA is SEQ ID NO: 3.
- 3. A vaccine composition comprising the recombinant mutant ASFV-G A9GL/AUK virus according to Claim 1 or Claim 2.
- 4. Use of a live attenuated ASFV-G A9GL/AUK virus in the préparation of a médicament for protecting swine against African Swine Fever Virus -Georgia 2007 isolate (ASFV-G), wherein the live attenuated ASFV-G A9GL/AUK vaccine comprises a recombinant mutant ASFV-G A9GL/AUK virus according to Claim 1 or Claim 2 in an amount effective to protect said swine from clinical ASF-G.
- 5. The use of Claim 4 wherein the amount effective to protect said swine from clinicalASF-G is a vaccine comprising 104 HAD50 to 105 6 HAD50 of ASFV-G A9GL/AUK virus.19131
- 6. A method of differentiating a mammal vaccinated with a live attenuated ASFV-G A9GL/AUK vaccine comprising a recombinant mutant ASFV-G A9GL/AUK according to Claim 1 from a non-vaccinated mammal infected with ASFV-G, said method comprising:a) obtaining a sample from a test mammal in need of being evaluated; andb) analyzing said sample for the presence of a gene normally présent in wild-type ASFV-G but not in the ASFV-G A9GL/AUK virus used for vaccinating said test mammal.
- 7. The method of Claim 6 wherein the step of analyzing said sample is performed with a PCR-based assay.
- 8. The method of Claim 6 wherein the step of analyzing is performed with an antibodydetecting assay or an ELISA.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US15/200407 | 2016-07-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA19132A true OA19132A (en) | 2020-01-31 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2017288932B2 (en) | A rationally developed african swine fever attenuated virus strain protects against challenge with parental virus georgia 2007 isolate | |
| US9528094B2 (en) | Attenuated African swine fever virus vaccine based in the deletion of MGF genes | |
| Gladue et al. | Deletion of the A137R gene from the pandemic strain of African swine fever virus attenuates the strain and offers protection against the virulent pandemic virus | |
| US11007263B2 (en) | Development of a novel live attenuated African Swine Fever vaccine based in the deletion of gene I177L | |
| O'Donnell et al. | Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge | |
| O'Donnell et al. | African swine fever virus Georgia isolate harboring deletions of MGF360 and MGF505 genes is attenuated in swine and confers protection against challenge with virulent parental virus | |
| O'Donnell et al. | African swine fever virus Georgia 2007 with a deletion of virulence-associated gene 9GL (B119L), when administered at low doses, leads to virus attenuation in swine and induces an effective protection against homologous challenge | |
| US9463234B2 (en) | Attenuated african swine fever virus strain induces protection against challenge with homologous virulent parental virus georgia 2007 isolate | |
| US9474797B1 (en) | African swine fever virus georgia strain adapted to efficiently grow in the vero cell line | |
| US20230124042A1 (en) | Vaccine against african swine fever virus infection | |
| US20200129614A1 (en) | H52 ibv vaccine with heterologous spike protein | |
| KR20230141819A (en) | Attenuated African swine fever virus and its use as a vaccine | |
| OA19132A (en) | A rationally developed African Swine Fever attenuated virus strain protects against challenge with parental virus Georgia 2007 isolate. | |
| NZ750036A (en) | A rationally developed african swine fever attenuated virus strain protects against challenge with parental virus georgia 2007 isolate | |
| JP2024500225A (en) | Genomic deletion in African swine fever vaccine that allows efficient growth in stable cell lines | |
| US11801296B2 (en) | Development of a novel live attenuated African swine fever vaccine based in the deletion of gene A137R | |
| Zhang et al. | Deletion of the L7L-L11L Genes Attenuates ASFV and Induces Protection against Homologous Challenge. Viruses 2021, 13, 255 |