史記署-用傳代細胞培養出來的安全和有效的非洲豬瘟弱毒疫苗.pdf

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1、Development of a Safe and Efficacious Live Attenuated Virus ASF Vaccine Using Cell Passage Technology利利用用細細胞胞傳傳代代技技術術研研制制安安全全有有效效的的A AS SF F減減毒毒活活疫疫苗苗Jishu Shi史史記記署署Professor of Vaccine Immunology疫苗免疫學教授Dr.Wayne and Hilda Appleton Endowed ProfessorDr.Wayne and Hilda Appleton 教授Director,Biologics Dev

2、elopment Module生物制品開發模塊總監Director,Center on Biologics Development and Evaluation生物制品開發和評價中心總監Director,U.S.-China Center for Animal Health美中動物衛生中心主任College of Veterinary Medicine獸醫學院Kansas State University,Manhattan,Kansas,USA美國堪薩斯州曼哈頓堪薩斯州立大學Center on Biologics 生生物物制制品品中中心心 Development&Evaluation開開發發

3、和和評評估估一帶一路國際生豬產業大會Presentation Outline匯匯報報大大綱綱My experience with experimental ASF vaccines我我使使用用實實驗驗性性A AS SF F疫疫苗苗的的經經驗驗VNUA-ASF-LAV 2 and VNUA-ASF-LAV3V VN NU UA A-A AS SF F-L LA AV V 2 2和和V VN NU UA A-A AS SP P-L LA AV V3 3Safety Evaluation安全性評估Efficacy Evaluation功效評估Analysis of Protective Immuni

4、ty 保護性免疫分析 一帶一路國際生豬產業大會32023 Beijing International ASF Workshop2 20 02 23 3年年北北京京國國際際A AS SF F研研討討會會ASF a serious transboundary animal disease with the potential for rapid international spreadA AS SF F是是一一種種嚴嚴重重的的跨跨界界動動物物疾疾病病，有有可可能能在在國國際際上上迅迅速速傳傳播播。Global distribution of ASF,2005-2023.2 20 00 05 5-2

5、 20 02 23 3年年A AS SF F的的全全球球分分布布。Map based on data from OIE World Animal Health Information system(https:/www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseaseoutbreakmaps?)and Global Disease Monitoring Reports(https:/www.swinehealth.org/global-disease-surveillance-reports/).Names of count

6、ries with ASF are given in the map.地圖基于世界動物衛生組織（OIE）世界動物衛生信息系統（https：/www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseaseoutbreakmaps？）和全球豬病監測報告（https：/www.swinehealth.org/global-disease-surveillance-reports/）的數據。地圖中給出了存在ASF的國家名稱。ASF is active in 40 countries around the worldA AS SF F活活躍

7、躍于于全全球球4 40 0個個國國家家一帶一路國際生豬產業大會Why is it difficult to prevent and control ASF?為為什什么么很很難難預預防防和和控控制制A AS SF F？1.The lack of a safe,efficacious,commercial and readily available vaccine缺乏安全的有效、商業化且易于獲得的疫苗2.The need for a suitable cell line for wildtype ASFV isolation and propagation,and for large-scale

8、LAV vaccine production野生型ASFV分離和繁殖以及大規模減毒活疫苗（LAV）生產對合適細胞系的需求一帶一路國際生豬產業大會African Swine Fever Virus:a complex and large virus非非洲洲豬豬瘟瘟病病毒毒：一一種種復復雜雜的的大大型型病病毒毒High consequence ASF etiological agent that causes high fever,loss of appetite,ataxia,depression and up to 100%mortality in pigs.一種危害性強的ASF病原體，可引起

9、豬高熱、食欲不振、共濟失調、抑郁和高達100%死亡率。First described in 1909 when it infected domestic pigs of European origin in Kenya.Confined in Africa in the long time.1909年首次描述，當時在肯尼亞感染了歐洲源家豬。長期流行于非洲地區。1950s,1960s outbreaks and eradication in 1990s by depopulation everywhere except in Africa.20 世紀 50 年代和 60 年代疫情暴發，20 世紀

10、 90 年代，非洲以外地區通過清群實現疫病凈化。Notable outbreaks:2007 Georgia,2018 China.值得注意的爆發：2007年格魯吉亞，2018年中國。Schematic illustration of ASFV virionA AS SF FV V病病毒毒粒粒子子的的示示意意圖圖Buan et al.,ImmunoInformatics.Vol 8,Dec 2022,100019.Buan 等人，免疫信息學。第 8 卷，2022 年 12 月，100019。一帶一路國際生豬產業大會19 ASF vaccine animal studies and 450 pi

11、gs 1 19 9項項A AS SF F疫疫苗苗動動物物研研究究和和4 45 50 0頭頭豬豬 2019-2024 ASF Viruses:A AS SF F 病病毒毒：Georgia 2007(Genotype 2)Georgia 2007（基因型2）Vietnam 2019(Genotype 2)Vietnam 2019（基因型2）OURT88/1(Genotype 1)OURT88/1（基因型1）Challenge dose:IM,0.1-100 HAD50攻毒劑量：肌內注射，0.1-100 HAD50Experimental Vaccines:實實驗驗性性疫疫苗苗：DNA/mRNA v

12、accinesDNA/mRNA疫苗Subunit vaccines亞單位疫苗Gene-deleted mutants基因缺失突變體Live attenuated viruses減減毒毒活活病病毒毒Objectives of Animal Studies動動物物研研究究的的目目的的ASFV virulence titrationASFV毒力滴定Vaccine safety疫苗安全性Vaccine efficacy疫苗效力Pigs豬豬：3-6 weeks old,female,from commercial farms 3-6周齡，母豬，來自商業豬場 Biosecurity Research In

13、stitute,Kansas State University堪堪薩薩斯斯州州立立大大學學生生物物安安全全研研究究所所一帶一路國際生豬產業大會BSL3 and BSL3Ag Research Facility B BS SL L3 3和和B BS SL L3 3A Ag g研研究究機機構構 at Manhattan,Kansas堪堪薩薩斯斯州州曼曼哈哈頓頓Dr.Quang Lam Truong at Vietnam National University of AgricultureQ Qu ua an ng g L La am m T Tr ru uo on ng g博博士士在在越越南南國國

14、立立農農業業大大學學Research Collaboration between Kansas State University and Vietnam National University of Agriculture 堪堪薩薩斯斯州州立立大大學學與與越越南南國國立立農農業業大大學學之之間間的的研研究究合合作作 VNUA-ASFV-LAVL2:KNB-LAV1V VN NU UA A-A AS SF FV V-L LA AV VL L2 2：K KN NB B-L LA AV V1 1VNUA-ASFV-LAVL3:KNB-LAV2V VN NU UA A-A AS SF FV V-L L

15、A AV VL L3 3：K KN NB B-L LA AV V2 2一帶一路國際生豬產業大會VNUA-ASFV-LAVL2:KNB-LAV1A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1,a Representative Strain of the Contemporary Pandemic African Swine Fever Virus一種細胞適應性減毒活疫苗候選株可保護豬免受同源株 VNUA-ASFV-05L1（當代流

16、行性非洲豬瘟病毒的代表株）的感染一帶一路國際生豬產業大會 Blood and Weight Collection血液和體重采集 Daily Temperature Monitoring using Thermal Microchips使用熱微芯片進行日常溫度監測Study Timeline研究時間表DPV0 疫苗接種第0天 ASF LAV VaccinationA AS SF F L LA AV V 疫疫苗苗接接種種15 days15天End of Study研研究究結結束束Challenge:ASFV Vietnam by IM injection攻攻毒毒：肌內注射非洲豬瘟病毒（ASFV）越

17、南株BRI ASF challenge phaseBRI A AS SF F攻毒階段D28/DPC0攻毒后第0天28 days28天D43/DPC15攻毒后第15天BRI ASFV LAV vaccination phaseBRI A AS SF FV V L LA AV V疫苗接種階段KNB-LAV1 evaluation by vaccination and challenge studies in pigs 通通過過豬豬的的疫疫苗苗接接種種和和攻攻毒毒研研究究評評估估K KN NB B-L LA AV V1 1 3-wk old pigs3 3周周齡齡仔仔豬豬1,000 HAD5010

18、HAD50VNUA-ASFV-LAVL2:KNB-LAV1V VN NU UA A-A AS SF FV V-L LA AV VL L2 2：K KN NB B-L LA AV V1 1一帶一路國際生豬產業大會KNB-LAV1 vaccinated pigs did not exhibit high temperatures during vaccination and challenge phases K KN NB B-L LA AV V1 1疫疫苗苗接接種種的的豬豬在在疫疫苗苗接接種種和和攻攻毒毒階階段段沒沒有有出出現現體體溫溫升升高高。一帶一路國際生豬產業大會KNB-LAV1 vacc

19、inated pigs continued to gain weight during vaccination and challenge phases 接接種種K KN NB B-L LA AV V1 1疫疫苗苗的的豬豬在在疫疫苗苗接接種種和和攻攻毒毒階階段段體體重重持持續續增增加加 一帶一路國際生豬產業大會KNB-LAV1 vaccinated pigs exhibited little or no viremia during vaccination and challenge phases K KN NB B-L LA AV V1 1疫疫苗苗接接種種的的豬豬在在疫疫苗苗接接種種和和攻攻

20、毒毒階階段段很很少少出出現現或或沒沒有有病病毒毒血血癥癥 Little or no viremia detected in sentinel pigs during vaccination phase.在疫苗接種階段，在哨兵豬中檢測到很少或沒有病毒血癥。Pig 193(DPV26)potentially non-ASF related(not tested).Pig 192(DPC9).193號豬（接種后第26天）可能與ASF無關（未檢測）。192號豬（攻毒后第9天）。一帶一路國際生豬產業大會132023 Beijing International ASF Workshop2 20 02 23

21、 3年年北北京京國國際際A AS SF F研研討討會會Cellular immune responses of pigs with low to high doses of KNB-LAV1低低至至高高劑劑量量K KN NB B-L LA AV V1 1對對豬豬的的細細胞胞免免疫疫應應答答 ELISpot testing the IFN-response in PBMC re-stimulation with wild-type ASFV at DPV28在在免免疫疫后后第第2 28 8天天用用A AS SF F野野毒毒株株再再刺刺激激P PB BM MC C時時檢檢測測I IF FN N-應

22、應答答的的E EL LI IS Sp po ot tPigs vaccinated with KNB-LAV1 developed cellular immunity against ASFV接接種種K KN NB B-L LA AV V1 1的的豬豬產產生生了了針針對對A AS SF FV V的的細細胞胞免免疫疫(EliSpot assay,IFN-producing cells/spot)(酶酶聯聯免免疫疫斑斑點點試試驗驗,產產I IF FN N-的的細細胞胞/斑斑塊塊)一帶一路國際生豬產業大會142023 Beijing International ASF Workshop2 20 02

23、 23 3年年北北京京國國際際A AS SF F研研討討會會Generation of ASF LAVs by genetically engineered deletions of specific virulence-associated genes通通過過基基因因工工程程敲敲除除特特定定毒毒力力相相關關基基因因生生成成A AS SF F L LA AV VReported virulence-associated genes deleted LAVs已已報報道道的的毒毒力力相相關關基基因因缺缺失失的的L LA AV Vs s一帶一路國際生豬產業大會15Comparison of KNB-

24、LAV1 with reported MGF gene-deleted LAVs from genotype II ASFVsK KN NB B-L LA AV V1 1與與已已報報道道的的基基因因I II I型型A AS SF FV V的的M MG GF F基基因因缺缺失失L LA AV V的的比比較較Comparison of known ASF viruses with MGF deletions 已已知知具具有有M MG GF F缺缺失失的的A AS SF F病病毒毒的的比比較較 一帶一路國際生豬產業大會162023 Beijing International ASF Workshop

25、2 20 02 23 3年年北北京京國國際際A AS SF F研研討討會會Genome characterization of VNUA-ASFV-LAVL2:KNB-LAV1 V VN NU UA A-A AS SF FV V-L LA AV VL L2 2：K KN NB B-L LA AV V1 1的的基基因因組組表表征征 Overview of Genome comparison of VNUA-ASFV-LAVL2 with parental virus VNUA-ASFV-05L1V VN NU UA A-A AS SF FV V-L LA AV VL L2 2與與親親代代病病毒毒

26、V VN NU UA A-A AS Sf fV V-0 05 5L L1 1的的基基因因組組比比較較概概述述一帶一路國際生豬產業大會172023 Beijing International ASF Workshop2 20 02 23 3年年北北京京國國際際A AS SF F研研討討會會Genome characterization of VNUA-ASFV-LAVL2:KNB-LAV1 V VN NU UA A-A AS SF FV V-L LA AV VL L2 2：K KN NB B-L LA AV V1 1的的基基因因組組表表征征 Mutations of VNUA-ASFV-LAVL

27、2 using the parental virus VNUA-ASFV-05L1 as a reference sequence使使用用親親代代病病毒毒V VN NU UA A-A AS SF FV V-0 05 5L L1 1作作為為參參考考序序列列對對V VN NU UA A-A AS Sf fV V-L LA AV VL L2 2進進行行突突變變一帶一路國際生豬產業大會Pigs vaccinated with KNB-LAV1:接種KNB-LAV1的豬：1.did not exhibit high temperatures during vaccination and challeng

28、e phases在疫苗接種和攻毒階段沒有表現出體溫升高2.continued to gain weight體重繼續增加3.developed significant ASFV-specific antibody and cellular immune responses產生了顯著的ASFV特異性抗體和細胞免疫應答4.exhibited little or no viremia during vaccination and challenge phases 在疫苗接種和攻毒階段很少或沒有病毒血癥 KNB-LAV1 safety and efficacy evaluation in pigs K

29、KN NB B-L LA AV V1 1在在豬豬中中的的安安全全性性和和效效力力評評價價 Summary KNB-LAV1總總結結 K KN NB B-L LA AV V1 1一帶一路國際生豬產業大會Presentation Outline匯匯報報大大綱綱My experience with experimental ASF vaccines我我使使用用實實驗驗性性A AS SF F疫疫苗苗的的經經驗驗VNUA-ASF-LAV 2 and VNUA-ASF-LAV3V VN NU UA A-A AS SF F-L LA AV V 2 2和和V VN NU UA A-A AS SP P-L LA

30、 AV V3 3Safety Evaluation安全性評估Efficacy Evaluation功效評估Analysis of Protective Immunity 保護性免疫分析 一帶一路國際生豬產業大會Growth characteristics and hemadsorption activity of VNUA ASFV-LAVL3(passage 50)and its parental virus VNUA-ASFV-L2V VN NU UA A A AS SF FV V-L LA AV VL L3 3（第第5 50 0代代）及及其其親親代代病病毒毒V VN NU UA A-A

31、AS SF FV V-L L2 2的的生生長長特特性性和和紅紅細細胞胞吸吸附附活活性性一帶一路國際生豬產業大會Rectal temperature and viremia in pigs inoculated with VNUA-ASFV-LAVL3 or parental VNUA-ASFV-L2 virus(before challenge)接接種種V VN NU UA A-A AS SF FV V-L LA AV VL L3 3或或親親代代V VN NU UA A-A AS S F FV V-L L2 2病病毒毒的的豬豬的的直直腸腸溫溫度度和和病病毒毒血血癥癥（攻攻毒毒前前）Vaccin

32、e virus was eliminated from the pigs by DPC 17-21截截至至攻攻毒毒后后第第1 17 7-2 21 1天天，疫疫苗苗病病毒毒從從豬豬中中消消除除，102 HAD50103 HAD50104 HAD50一帶一路國際生豬產業大會Rectal temperature and viremia in pigs following challenge with virulent wild-type VNUA-ASFV-05L1 strain(genotype II)用用野野生生型型V VN NU UA A-A AS SF FV V-0 05 5L L1 1強強

33、毒毒株株（基基因因I II I型型）攻攻毒毒后后豬豬的的直直腸腸溫溫度度和和病病毒毒血血癥癥一帶一路國際生豬產業大會Gross pathology in organs of vaccinated and non-vaccinated pigs post-challenge at necropsy攻攻毒毒后后尸尸檢檢時時接接種種疫疫苗苗和和未未接接種種疫疫苗苗豬豬器器官官的的大大體體病病理理學學Control pigs對照豬Vaccinated pigs疫苗接種豬一帶一路國際生豬產業大會Long-term protection conferred by a single vaccination

34、of VNUA-ASFV-LAVL3 in experimental pigs單單次次接接種種V VN NU UA A-A AS SF FV V-L LA AV VL L3 3疫疫苗苗對對實實驗驗豬豬的的長長期期保保護護作作用用一帶一路國際生豬產業大會3-wk LAV pigs3 3周周齡齡L LA AV V豬豬 6-wk LAV pigs 6 6周周齡齡L LA AV V豬豬 103 HAD50104 HAD50105 HAD50VNUA-ASFV-LAVL3 induces both robust humoral and cellular immunity,leading to long-

35、term protection against highly virulent genotype II ASFV challengeV VN NU UA A-A AS SF FV V-L LA AV VL L3 3可可誘誘導導強強大大的的體體液液和和細細胞胞免免疫疫，從從而而對對高高毒毒力力基基因因I II I型型A AS SF FV V攻攻毒毒產產生生長長期期保保護護作作用用一帶一路國際生豬產業大會130,00060,00090,000120,000150,000179,678mutation突變Deletion刪除Insertion插入285L,MGF110_8L-14L,100_1R,3

36、60_4L,6L,X69R 2 28 85 5L L、M MG GF F1 11 10 0_ _8 8L L-1 14 4L L、1 10 00 0_ _1 1R R、3 36 60 0_ _4 4L L、6 6L L、X X6 69 9R R CD2VI177LVNUA-ASFV-LAVL3 genome schematic illustration(Compared to the parental strain)V VN NU UA A-A AS SF FV V-L LA AV VL L3 3基基因因組組示示意意圖圖（與與親親代代毒毒株株相相比比）一帶一路國際生豬產業大會Mutations

37、 in CD2v gene caused disruption of CD2v protein of VNUA-ASFV-LAVL3C CD D2 2v v基基因因突突變變導導致致V VN NU UA A-A AS SF FV V-L LA AV VL L3 3的的C CD D2 2v v蛋蛋白白破破壞壞一帶一路國際生豬產業大會Conclusions結結論論The cell-adapted non-HAD VNUA-ASFV-LAVL3 can replicate efficiently in commercially available swine macrophage cell line

38、3D4/21.細胞適應的非HAD VNUA-ASFV-LAVL3可以在商業豬巨噬細胞系3D4/21中有效復制。A single nucleotide substitution and two single nucleotide deletions in the EP402R gene of VNUA-ASFV-LAVL3,leading to frameshift variant in the CD2v protein,might be responsible for the loss of hemadsorption with VNUA-ASFV-LAVL3 strain.VNUA-ASFV

39、-LAVL3的EP402R基因中有一個單核苷酸替換和兩個單核苷酸缺失，導致CD2v蛋白移碼變異，這可能是VNUA-ASfv-LAVL3毒株紅細胞吸附喪失的原因。VNUA-ASFV-LAVL3 is a safe and efficacious vaccine against genotype II ASFV.VNUA-ASFV-LAVL3是一種安全有效的抗基因II型ASFV疫苗。VNUA-ASFV-LAVL3 induces both robust humoral and cellular immunity,leading to long-term protection against hig

40、hly virulent genotype II ASFV challenge.VNUA-ASFV-LAVL3可誘導強大的體液免疫和細胞免疫，從而對高毒力基因II型ASFV攻毒產生長期保護。一帶一路國際生豬產業大會What an ideal(safe and efficacious)ASF vaccine should look like?理理想想（安安全全有有效效）的的A AS SF F疫疫苗苗應應該該是是什什么么樣樣子子的的？Classical swine fever(CSF)vaccines豬豬瘟瘟（C CS SF F）疫疫苗苗Modified live virus(MLV)vaccin

41、es:C-strain (DIVA compatible)改改良良活活病病毒毒（M ML LV V）疫疫苗苗：C C株株（D DI IV VA A兼兼容容）Subunit:E2 based subunit vaccines(DIVA)亞亞單單位位：E E2 2亞亞單單位位疫疫苗苗（D DI IV VA A）一帶一路國際生豬產業大會When can we have an ideal MLV ASF vaccine?我我們們何何時時能能獲獲得得理理想想的的M ML LV V A AS SF F疫疫苗苗？/Yes/是是一帶一路國際生豬產業大會Acknowledgement致致謝謝Funding So

42、urces資資金金來來源源(2012-2023)NBAF Transition FundNBAF過渡基金BioApp,DingChiAnimal Health Industry 動物健康產業 USDA ARS,USDA FAS美國農業部ARS、美國農業部FASUSDA NIFA,DHSDr.Rachel MaderaR Ra ac ch he el l M Ma ad de er ra a博博士士Dr.Lihua WangL Li ih hu ua a W Wa an ng g博博士士Yuzhen LiAidan Craig艾艾丹丹克克雷雷格格Nina MuroJimena KilianHea

43、ther George希希瑟瑟喬喬治治Dr.Matias-Ferreyra 馬蒂亞斯-費雷拉博士 Dr.Quang Lam Truong at VNUAQuang Lam Truong博士在VNUADr.Manuel Borca曼紐爾博卡博士Dr.Douglas Gladue道格拉斯格拉杜博士Dr.Cyril Gay西里爾蓋伊博士USDA ARS 美美國國農農業業部部A AR RS S Dr.Linda DixonLinda Dixon博士Pirbright InstituteP Pi ir rb br ri ig gh ht t研研究究所所Isabelle KlossIan Hallenbeck伊伊恩恩哈哈倫倫貝貝克克Dr.EunJu SohnDr.Hyangju KangDr.Ju Hun KimBioAppDr.Ningning MaDr.馬寧寧Dr.Jinan HuangDingChi丁丁馳馳Dr.Jamie RetallickXirui Zhang張張喜喜瑞瑞一帶一路國際生豬產業大會