New developments in vaccines against Better lives through livestock African swine fever Lucilla Steinaa Principal Scientist Animal and Human Health Program, ILRI International Veterinary Vaccinology Network Webinar 30 November 2021 2 Spread of African Swine Fever Virus 2018 2018 2018 1957 1960 2007 Dixon et al., Antiviral Research 2019 3 African Swine Fever Status (2016-2020) 4 African Swine Fever in Africa • Large DNA virus, Asfaviridae family • Approximately 160 genes, number depending on isolate. • 2 genotypes present in China, one in Europe. • ASFV present in about 26 African countries. • All 24 genotypes are present in Africa. • There is a wildlife reservoir: warthogs and bush pigs. • Wild boars are susceptible. • Soft ticks of the genus Ornithodores are involved in transmission of ASFV. From “Encyclopedia of Virology” Chap.: African swine fever” by L.Dixon and D. Chapman. 2008 5 CAM/1/02 CAM/4/85 GHA/1/02 LIS/60 NIG/1/01 ANG/70 I 91 IC/2/96 BEN/1/97 Genotype I SPEC/205 SPEC/209 Mkuzi/79 MOZ/1/02 MOZ/1/05 LUS1/93 II MOZ/2/02 MOZ/1/03 p72 gene 57 RSA/5/95SPEC/257 RSA/3/03 III RSA/Warmbaths MOZ/1979 MAL/1/02 Genotype II,III, IV, V, MOZ/1960 V Tengani/62 VI, VII, XIX, XX, XIII MOZ/8/94 SPEC265 VI MOZ/1/94 74 RSA/3/04/ RSA/Warthog IV RSA/1/99/W RSA/2/96 RSA/2/03 XIX RSA/3/96 RSA/1/04 Lillie XX RSA/1/95 RSA/1/03 XIII RSA/4/95 RSA/1/98 VII Genotype VIII, XI, XII, XIII, SPEC/154 NYA/12 XIV XV, XVI, XIV TAN/2/03 XVI 79 TAN/1/03 TAN/1/01 XV SUM/14/11 XIII MZI/1/92 XII KAB/6/2 XI MCH/1/89 MCH/3/89 BAN/1/91 DED/1/89 Genotype IX, X LIL/20/2 DOWA VIII CHJ/1/89 KLI/2/88 PHW/1/88 TMB/1/89 UGA/1/95 IX KEN/5/01 100 BUR/1/84 BUR/1/90 X Kenya/50 0.005 Neighbor-Joining tree depicting the p72 gene relationships and geographical distribution of the major ASFV genotypes Contribution: Livio Heath (ARC-OVI) TOWARDS A LIVE ATTENUATED VACCINE FOR AFRICAN SWINE FEVER 7 Vaccine Candidates 8 Vaccine Candidates • Safety • Efficacy • Different dose studies for some vaccine • High level of protection, 100 % in many candidates. cases in various doses • Very different clinical readout system, some use • Under optimal timing, 4 weeks post clinical score systems with many parameters immunization (King 2011 and Galindo-Cardiel 2013), others • Duration of immunity ? use single parameters, e.g., fever. • Route of immunization • Initially: intramuscularly • Orally route became interesting because of wild boar • Less viremia using orally route 9 ILRI ASFV Vaccine Activities • Live vaccine (CRISPR/Cas9 deletions) and synthetic approach • Deletion of genes for attenuation • Testing in established animal model • Subunit vaccine – activities • Screening of antigens • Viral vectors as delivery 10 Isolated Virus • Kenya 1033 (genotype IX) isolated by ILRI and DVS Kenya. • Genotype IX and X are especially circulation in Eastern Africa. • Isolated from a zone with outbreaks. • Used as the challenging virus in the animal model • Used as backbone for deletion of genes to generate attenuated viruses. Gallardo C et al. A.J. Biotech 2011 Onzere C. et al. Virus Genes 2018 11 ASFV Kenya 1033 – Virus Batch for Challenge • This virus is very similar to the other genotype IX and X viruses. • Animal model was set up. Different doses were tested. 5 animal per group, intramuscular injection. Survival - Groups Body temperature - Groups Clinical score - Groups 15 2 2 2 100 10 10 10 42 103 103 103 10 104 104 104 5 4050 10 105 105 5 38 0 0 0 2 4 6 8 10 0 2 4 6 8 0 2 4 6 8 10 Day after challenge Day after challenge Day after challenge Scoring system: Galindo-Cardiel 2013 Percent survival Body temperature Clinical score 12 Genomic Stability and Production Cell Lines Problems with instability of genomes in cell lines Progress on production cell lines ZMAC – pig macrophage cell line MA-104 cell line (Green monkey kidney epithelial cell line) 13 Virulence of WSL Adapted WT-Virus • WSL (from FLI) is a fetal wild boar lung cell line, not immortalized. • ASFV Kenya 1033 was adapted to WSL (20+ passages) • 102 TCID50 was chosen to test if the virus grown in WSL cells was still lethalVirus batch for challenge experiments 25 100 20 Challenge with wild type virus. 15 50 Open circles: WSL cell line grown 10 virus , Solid squares: Macrophage 5 0 grown virus 0 2 4 6 8 10 0 0 1 2 3 4 5 6 7 8 Days post infection Days post infection Scoring system: King et al. 2011 Probability of Survival Clinical score 14 Titers of ASFV Ken-1033 in WSL 1.00E+10 1.00E+09 1.00E+08 1V.00iE+r07us batch for challenge experiments 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Macrophage grown ASFV - 4 days WSL grown ASFV - 4 days MOI 0.1 MOI 0.25 MOI 1 MOI 2.5 MOI 5 15 CRISPR-Cas Editing of African Swine Fever Virus Transfection with guide RNA and GFP donor DNA Infect with WT-virus ➢ Directly ASFV gene modification on Stable CAS transfected replicating virus LHA RHA cell line (WSL) inside cells Check for GFP insert with PCR over junction LHA RHA GFP in ASF genome Cloning of cells with fluorescent virus Constructed 7-10 different viruses 16 Synthetic Construction of African Swine Fever Virus Virus Genome Validated Parts + Isolate genome and rescue virus + Modified Parts ➢ Capacity to efficiently perform genome-wide changes in the virus genome in a combinatorial manner to understand virus biology. ➢ Capacity to produce clinically-relevant viruses without extensive passaging in tissue culture. ➢ Streamlines process to generate various designer vaccine candidates and oncolytic viruses. CD2v 17 First Viruses: Experimental Setup Immunomodulatory molecule promoting apoptosis of lymphocytes. A238L Mimic NFκB subunit, inhibits NFκB activity, which is Immunisation (1 injection) Challenge crucial in the pro-inflammatory response. 104 ASF1033_∆CD2v (9x) 102 ASF1033 (8x) 104 ASF1033_∆CD2v∆A238L (9x) 102 ASF1033 (8x) PBS (9x) 102 ASF1033 (8x) Quarantine Clinical scoring Clinical scoring Day -21 0 31 51 18 Clinical Scores After Immunization AASSFF1013033_3_C∆DC2Dv2v AASSFF11003333__∆CCDD2v2vA∆2A3283L8L PBS PBS 20 20 20 15 15 15 10 10 10 5 5 5 0 0 0 0 10 20 30 0 10 20 30 0 10 20 30 DPI DPI DPI Weight gain Post-immunisation 7.5 15 ASF1033_CD2v ASF1033_CD2v ASF1033_CD2vA238L 5.0 ASF1033_CD2vA238L10 PBS PBS 2.5 5 0.0 1 2 3 4 0 -2.5 Week 0 10 20 30 DPI Clincal sore Kg Clinical score Clinical score Clincal score 19 Clinical Scores After Challenge ASF1033_CD2v ASF1033_CD2vA238L PBS ASF1033_∆CD2v ASF1033_∆CD2v∆A238L PBS 20 20 20 15 15 15 10 10 10 5 5 5 0 0 0 0 7 14 21 0 7 14 21 0 7 14 21 DPC DPC DPC Challenge Clinical scores Challenge 20 ASF1033_CD2v 20 ASF1033_CD2v 15 ASF1033_CD2vA238L 15 ASF1033_CD2vA238L PBS PBS 10 10 5 5 0 0 0 7 14 21 0 7 14 21 DPC DPC Clincal score Clincal score Clinical score Clinical score Clinical score 20 Survival Plot Survival proportions 100 ASF1033_CD2v ASF1033_CD2vA238L PBS 50 IFNγ-ELISpot D28 1000 ASF1033_CD2v 0 800 ASF1033_CD2vA238L 0 7 14 21 PBS 600 DPC 400 200 0 Probability of Survival SFU/ million cells medium WT medium WT medium WT 21 Conclusion • ∆CD2v is more efficient than the double knockout but less attenuated. 87.5% protection versus 50%. • ∆A238L seems to add to the attenuation, but with a loss in ability to protect. Controls 18 16 ΔA238L-only 14 12 10 8 6 4 2 0 0 5 10 15 20 25 DPI Clinical score 22 Second viruses: Experimental Setup Immunisation (1 injection) Challenge 103 ASF1033_∆X (7x) 102 ASF1033 _∆X (6x) 103 ASF1033_∆Y (7x) 102 ASF1033 _∆Y (6x) PBS (7x) 102 ASF1033 (6x) Quarantine Clinical scoring Clinical scoring Day -21 0 2381 51 23 New Gene-Deleted Viruses A ll g r o u p s 2 5 IEFNl isγp-EoLt IDS2p8o_t2 Dx2180 ^ 5 Group1 1 0 0 0 G ro u p 1 2 0 Group2 8 0 0 G ro u p 2 Immunization Challenge Group3 G ro u p 36 0 0 1 5 4 0 0 2 0 0 1 0 0 5 -2 0 0 A .1I 0 A D _ _n E I o M OM C 0 T W V F S A D a y s p o s t in fe c tio n G r o u p 2 G r o u p 1 2 5 2 5 Remaining data: 2 0 2 0 Viremia data 1 5 1 5 Immunization Challenge Immunization Challenge 1 0 1 0 Antibody titers 5 5 0 0 PM data 0 1 0 2 0 3 0 4 0 5 0 0 1 0 2 0 3 0 4 0 5 0 Days post infection Days post infection D P I C lin ic a l s c o re s (m e a n ) 0 1 2 3 4 5 6 7 8 C lin ic a l s c o re (a v e ra g e ) 9 10 11 12 13 14 15 16 17 18 19 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 C lin ic a l s c o re (a v ) 3 6 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 S F U /1 0 ^ 6 P B M C 24 Acknowledgements ILRI ∆CD2v virus / WT-virus Collaborators Hussein Abkallo Friedrich Loeffler Institute Sanjay Vashee, Hanneke Hemmink Gunther Keil J. Craig Venter Institute Nicholas Svitek Raquel Portugal Jeremiah Khayumba Sandra Blome Walter Fuchs, Anna Lacasta Friedrich Loeffler Institute Elias Awino ILRI Rosemary Saya Richard Bishop, now WSU Bernard Odour Edward Okoth Emanuel Khazalwa Lucilla Steinaa THANK YOU