Current approaches for African swine fever virus vaccine development
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Current approaches for African swine fever virus vaccine development

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Presented by Linda K. Dixon at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011

Presented by Linda K. Dixon at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011

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Current approaches for African swine fever virus vaccine development Current approaches for African swine fever virus vaccine development Presentation Transcript

  • Current Approaches for African Swine Fever Virus Vaccine Development Linda K. Dixon11 Institute for Animal Health, Pirbright Laboratory, UK
  • IAH Resources for ASFV Research•High containment (BSL4) laboratory, largeanimal facilities and insectary•OIE Reference Lab for ASFV•Large collection of ASFV strains andreagents•Interdisciplinary research programmes•3 lines of inbred pigs, colonies ofOrnithodoros ticks New CL4 Laboratory Complex being built at IAH Pirbright
  • African swine fever virus• Large double-stranded DNA virus, genome length 170-190 kbp• Only member of virus family the Asfarviridae• Replicates in the cytoplasm – similar strategy to Poxviruses• Virus particle contains RNA polymerase and other enzymes needed to start replication cycle – virus DNA is not infectious• Encodes about 151-167 genes including enzymes required for replication and transcription of the virus genome• Many genes ( ~1/3) are not essential for virus replication in cells but play an important role in virus survival and transmission• Replicates mainly in macrophages in vivo• No vaccineInstitute for Animal Health
  • Nucleo-cytoplasmic large DNA virus superfamily
  • ASFV structure •ASFV virions have a complex multilayer structure •More than 50 proteins are present •Extracellular and intracellular mature virions are both infectious a) schematic showing layers in extracellular virions b) extracellular virions budding c) and d) intracellular virus factories showing immature (IM) and mature (M) virions c) chemical fixation, d) high pressure freezingPippa Hawes IAH 200 nm
  • Virus Particle P72 cD2V p22p54 Proteins on surface of extracellular and intracellular virus particle targets for antibody mediated protection B438L
  • Virus Genome 160-175 genes Many not essential for replication 182 kbpBenin 97/1 complete genome Replication MGF100 MGF360 Structural P22 MGF110 unknown evasion MGF505/530
  • Genes involved in immune evasion/virulenceInhibitors of host signalling pathways that blocktranscription of host immunomodulatory genes A238L, broad inhibition of host gene transcription. - Inhibitors of IFN -Inhibitor of Toll-like receptors TLR 3 and 4 Adhesion proteins CD2v, causes binding of infected cells and virus particles to red blood cells, impairs lymphocyte proliferation C-type lectin -resembles NK cell inhibitoryreceptors Apoptosis inhibitors – IAP and Bcl2 homologuesComparison of sequences of non-pathogenicand pathogenic strains
  • Red Blood Cells bound to ASFV Extracellular virus particles infected macrophages bound to Red Blood Cells RBC V V V RBC ASFV infected macrophage “Hides” virus particles and infected cellsCourtesy Sharon Brookes
  • Pathogenesis• Highly virulent isolates ~100% death of pigs within 5 to 12 days. – High viraemia (> 10 8 ) Apoptosis of lymphocytes Damage to endothelial cells lining blood vesicles, disseminated intravascular coagulation, haemorrhage• Moderately virulent isolates cause death of 30 to 50 % of pigs. - Disease similar to highly virulent isolates but survivors tend to have lower viraemia (10 4-6). Virus persists in recovered pigs• Low virulence isolates. Very few deaths. - Occasional low viraemia 10 2-3 and fever. Virus in tissues. Persistent infection in pigs.• Pigs which recover from infection are protected against challenge with lethal dose of related virulent viruses• Low virulence isolates provide good model for understanding protection
  • Non-pathogenic OurT88/3 has deletions and insertions compared to highly pathogenic Benin97/1 isolateLeft end MGF110 MGF 530 3FR, NRMGF110 Benin 97/1 MGF 360 3CL, DL, EL MGF 360 3HL, IL, LL
  • Summary: Genome comparisons Benin 97/1 (highly pathogenic) compared to OUR T88/3 (non-pathogenic)• Gene deletions at left end of OURT88/3 genome include members of MGF360 (6 copies) and MGF530 (2 copies) implicated in virulence, cell tropism and IFN induction• CD2v and C-type lectin genes interrupted in OURT88/3. CD2v implicated in impairing lymphocyte activation• MGF 300 (1 copy) and MGF 110 (2 copies) in Benin not OUR T88/3• MGF 110 (4 copies) and 4 other ORFs in OUR T88/3 not Benin.• Conserved ORFs encode proteins with 98 to 100% identity.• Two ORFs encode proteins with variable numbers of tandem repeats.
  • ASFV Multigene families• 5 Multigene Families (MGFs) – A set of genes derived by duplication of an ancestral gene followed by independent mutational events resulting in a series of independent genes• Constitute ~17% - 25% of the coding capacity• Lack similarity to other known genes, functions unknown• Vary in gene number between ASFV isolates: – MGF 100: 2-3 genes per genome – MGF 110: 5-13 genes per genome – MGF 300: 3-4 genes per genome – MGF 360: 11-19 genes per genome – MGF 530: 8-10 genes per genome
  • Deletion of MGF360 and MGF530 reduces virus growth in macrophages and virulence in pigs macrophage virulence in tick IFN replication pigs replication induction + + + - + NT + NT + NT + NT + NT NT 102-103 + NT + 102-103 102-103 Note these MGF 360 and 530 genes are also deleted from non-pathogenic isolate (Chapman et al., 2008) Zsak et al., 2001, Neilan et al., 2002, Afonso et al, Burrage et al.,2004
  • Prospects for vaccine development• Survivors of ASF can resist challenge by related virulent viruses (eg De Tray 1957, Malmquist 1963, Handy and Dardiri 1983) - therefore prospects for ASFV vaccine development are good
  • Obstacles to ASFV vaccine development• Inactivated ASF virions do not induce protection• Serially passaged ASFV vaccine strain used in Portugal and Spain in 1960s caused post-vaccination reactions in 128,684 of 550,000 vaccinated -Loss in confidence and need for extensive tests of vaccine emphasised• Complexity of virus (~160-175 genes encoded. Virus particles contain > 50 proteins in several concentric layers)• Neutralising antibodies are not effective• Genetic complexity. Many virus genotypes (22) have been defined by sequence of the gene encoding the major capsid protein.
  • However -• Highest ASFV diversity is in natural hosts (warthogs and O. moubata ticks) in E and S Africa. Spread of genotypes to domestic pigs is limited and in some endemic areas a single genotype is circulating• In addition cross-protection can be induced between genotypes (King et al., 2011)• ASFV is a large DNA virus with more accurate replication than RNA viruses. This results in a relatively stable genome.
  • Pigs can be protected:• Survivors of ASF can resist challenge by related virulent viruses (eg De Tray 1957, Malmquist 1963, handy and Dardiri 1983) .Pigs are protected when:• Inoculated with viruses attenuated by passage in tissue culture, eg E75CV (Ruiz Gonzalvo et al., 1986, Gomez-Puertas et al., 1998)• Inoculated with natural low virulence isolates, eg NHP68, OurT88/3 ( Leitao et al., 2001, Boinas et al., 2004, Denyer et al., 2006)- Low sporadic or no viraemia detected, protection close to 100%.• Inoculated with recombinant virus with single genes deleted (Lewis et al., 2000, Neilan et al., 2004).- Viraemia 10 3-6 over ~20 days. High percentage protection
  • Understanding mechanisms of protection• Identification of correlates of protection for vaccine development• Identification of protective immune mechanisms directs strategies for vaccine development
  • Mechanisms of protection induced byattenuated viruses: A role for CD8+ T cells• CD8+ T cells are necessary. Depletion of CD8+ T cells abrogates protection induced by OURT88/3 (Oura et al., 2004)• Protection correlates with frequency of ASF specific IFN-gamma producing memory T cells• Ability of different virus isolates to stimulate lymphocytes from OURT88/3 immune pigs correlates with cross-protection (King, et al., Vaccine 2011)• Key virus antigens involved in inducing immunity mediated by T cells not defined.
  • Mechanisms of protection induced byattenuated viruses: The role of antibodies• Pigs can be protected by passive transfer of antibodies from immune pigs (Onisk et al., 1994). Higher viraemia observed than in pigs protected by attenuated virus• Mechanism by which antibodies protect: - pre virus entry (neutralisation), targets identified p54 (E183L), p30 (CP204L), p72 (B646L) - post virus entry (infection inhibition), mechanism and targets not known• Inhibition of infection in vitro by immune serum correlates with cross-protection observed in vivo against different isolates
  • Experimental vaccination with attenuated ASFV strain OURT88/3 OURT88/1 i.m. 10 4 virulent genotype I OURT88/3 i.m. 10 4 Benin 97/1 i.m. 10 4 or OURT88/3 non-virulent genotype I virulent genotype IBloodsampling – Temperature andserum and clinical scoreswhole blood 0 7 14 21 28 36 41 49 d56 Termination of experiment Introduction of non-immune pigs
  • ASFV viraemia and clinical score in vaccinated compared to control pigs 2.5 x 107 Copy number per ml OURT88/3 OURT88/1 Benin 97/1 VR89 2.0 x 107 VR90 ASFV detected only VR92 1.5 x 107 from non-immune VR97 pigs VR98 VR99 1.0 x 107 VS00 5.0 x 106Days postOURT88/3 0inoculation 20 18 VR89 VR90 Clinical score Benin 97/1 16 VR92 14 VR97 12 Non-immune pigs VR98 VR99 10 VS00 8 6 4 Days post 2 Benin 97/1 0 1 0 1 2 3 4 5 6 7 8 9 10 challenge
  • IFN-γ ELISPOT 80000 Proliferation assay 1000 8 A VR89 D VR89 800 ● 60000 6 ● ● ● 600 ● ● 40000 4 400 ● 20000 2 ● 200 ●ASFV specific IFN-γ production frequency per 0 ▲ ▲ ▲ ▲ ▲ ▲ ▲ 0 0 W-0 W-1 W-2 W-3 W-4 W-5 W-6 ● W-8 0 1 2 3 4 5 6 8 0 1 2 3 4 5 6 8 104) 3H-TdR uptake (∆ cpm X 120000 1000 12 B VR90 ● 100000 10 E VR90 ● 800 ● ● ● ● 8 ● 80000 600 ● ● 60000 6 400 40000 4 200 20000 2 ● 0 ● ● ▲ ▲ ▲ ▲ ▲ ▲ ▲ 0 0 W-0 W-1 W-2 ● W-3 W-4 W-5 W-6 W-8 0 1 2 3 4 5 6 8 0 1 2 3 4 5 6 8106 PBMC 1000 100000 10 800 C VR92 ● 80000 8 F VR92 ● 600 ● 6 60000 ● ● 400 4 40000 ● 200 ● ● 20000 2 ● ▲ ● ● 0 ● 0 ▲ 1 ▲ 2 ▲ 3 4 ▲ 5 ▲ 6 ▲ 8 0 0 W-0 0 W-1 1 ● 2 W-2 W-3 3 W-4 4 5 W-5 W-6 6 W-8 8 Week post first vaccination Frequency of IFNγ producing cells increases after 1st immunisation and is boosted after 2nd
  • Anti- ASFV p72 antibody responses Exp 2 14.00 A 12.00 Anti-ASFV antibody 10.00 1803 8.00 1826 titre 6.00 1834 4.00 1845 2.00 0.00 0 10 20 30 40 50 60 14.00 B Anti-ASFV antibody titre 12.00 1809 10.00 1811 8.00 1829 6.00 1837 4.00 2.00 1844 0.00 1822 0 10 20 30 40 50 60 Days post 1st immunisationAnti-p72 response rises to day 20 and is boosted by 2nd immunisation. Infectioninhibition assays showed low inhibition of infection in vitro (up to 10 2 )
  • Recognition of diverse strains of ASFV by lymphocytes from OURT immune pigs correlates with protection Cross-reactivity of OURT88/3 immune pigs PBMC to Cross reactivity to OURT88/3 other ASFV isolates : IFN-g ELISPOT Assay 140 120 OURT88/3 OURT88/3 Type I % Cross-reactivity Benin 97 Benin -5 Type 1 % cross-reactivity 100 Lisbon 57 Lisbon Type 1 80 Malawi malawi Type VIII 60 Malta 78 malta Type I 40 Uganda uganda Type X 20 OURT88/1 OURT 1-6 Type I 0 Pig number VR89 VR90 VR92Good correlation between IFN-γ cross-reactivity and cross-protection • OURT88/3, OURT88/1 immune pigs protected against virulent African isolates ASFV Benin 97 and Uganda challenge. No cross-protection to Malawi, only partial protection to Lisbon 57
  • Comparison of complete genomes of Georgia 2007/1 isolate with other ASFV isolates Kenya 69 Malawi 88 Georgia 2007/1 Mkuzi 79 OURT88/3 BA71V W. Africa Benin 97/1 Europe E70 Tengani 62 Warmbaths E. and S. Pr 96/4 Africa 0.004 WarthogComparison of the concatenated sequences of 125 conserved genes (~40,000 amino acids)shows the Georgia 2007 isolate is in the same clade as those from Europe and W. Africabut more distantly related -Chapman et al., Emerging Infectious Diseases 2011
  • Survival of pigs challenged with ASFV isolates form genotype I and X Challenge of immune 100 Immune - Benin 80 pigs with different ASFV Percent survival 60 isolates: % survival 40 Exp 1 Exp 1 IAH, UK 20 Benin Exp 2 ANSES, France –SPF 0 0 5 10 Exp 3 ANSES, France 100 Days post challenge Exps 1 and 3 100% 80 Immune - Uganda immunised pigs survived Percent survival Immune - Benin 60 challenge with genotype 1 40 Benin Exp 2 Benin 97/1 20 Exp 2 60% Uganda 0 0 5 10 immunised pigs survived Days post challenge challenge with genotype 1 100 OURT88/3 - OURT88/1 - Benin Benin 97/1 and 100% OURT88/3 x 2 - Benin 80 Percent survival genotype X Uganda 60 Some adverse effects of 40 Exp 3 20 immunisation in experiments 0 Benin in France 0 5 10 15 20 Days post challenge
  • Challenges for attenuated vaccines• Safety concerns about release of replicating virus vaccine• High containment required for production• Optimised cell culture required for growth of vaccine strain• Current strains may not be sufficiently attenuated• Additional genes involved in virulence deleted from attenuated strains
  • Virus Genome 160-175 genes Many not essential for replication 182 kbpBenin 97/1 complete genome Replication MGF100 MGF360 Structural P22 MGF110 unknown evasion MGF505/530
  • Effect of ASFV gene deletionsGene Function Effect on Effect on Conserved in virulence replication in isolates culturedUTPase, Nucleotide Reduced Reduced YesThymidine metabolism replication inKinase macrophages9GL Virion Reduced Reduced Yes morphogenesisMGF Unknown Reduced Reduced No360/530 IFN induction?CD2V Binding red Delayed No effect No blood cells, dissemination lymphocyte no reduction function in mortalityDP71L PP1 regulator Can reduce No effect Present as (short form) long or short form
  • Effect of ASFV gene deletionsGene Function Effect on Effect on Conserved in virulence replication isolatesA238L Inhibitor of None None Yes host transcriptionC-Type lectin Inhibition of None None No MHC class I presentationIAP Apoptosis None None Yes inhibitionUK Unknown Reduced None Yes
  • Subunit vaccines• Partial protection achieved with recombinant proteins expressed in baculovirus: - a mixture of proteins p30 and p54 (Gomez-Puertas et al., 1996) – NB Neilan et al., 2004 reported no protection - CD2-like protein (or haemmaglutinin) (Ruiz-Gonzalvo et al., 1999)• Delay in onset of disease signs and viraemia, some pigs recover from infection and clear virus
  • Challenges for subunit vaccines• Identification of additional protective antigens especially dominant antigens recognised by CD8+T cells• Identification of vaccine delivery systems for pigs to induce cell-mediated and antibody responses eg host restricted virus vector such as swinepox or avipox
  • Rapid vaccine development platform• Collaboration Kathy Sykes, Bert Jacobs, Biodesign Institute, Arizona State University, IAH Pirbright UK• Genome wide screen of ORFs encoded by Georgia 2007 ASFV isolate to rank proteins for induction of cell mediated and antibody responses in pigs• Genes delivered in pools of 20- 40 to pigs by DNA/prime recombinant vaccinia virus boost• Antibody and cell mediated immune responses to individual antigens measured using individual in vitro translated proteins• Test smaller pools “best” antigens for ability to protect pigs against lethal ASFV challenge
  • Strategy for ASFV Library Construction
  • Genome wide screen for protective ASFV antigens Collaboration IAH- Biodesign Institute, Arizona State UniversityImmunize pigs withexpression libraries inpools by DNA prime Assay sera, PBMC, RNA for immune responsesrecombinant vacciniavirus boost Sort and Rank all ORFs Ab Iso- Th1 Th2 Cyto- type kine ORF10 ORF5 ORF69 ORF50 ORF100Test in Pig Select antigens to ORF113 ORF811 ORF98 ORF63 ORF39Challenge- test . . . . . . . . . .Protection . . . . .Assays . . . . . . . . . . . . . . . . . . . .
  • Proteome-scale protein production and purificationT7 RBS ATG TRX ORF His Term In vitro synthesis of proteins Linear DNAs for in vitro transcription/translation Magnetic beads for capture and purification proteins
  • Challenge/protection experiments1. Pool top antigens from each bin and immunize pigs with these pools of antigens by DNA prime and recombinant vaccinia virus boost. Immunize Survival readout Challenge ?2. Pool top 5-10 antigens from positive bins, and immunize pigs.3. Re-test and validate vaccine candidates
  • 3H-TdR uptake, cpm 0 100 200 300 400 500 600 700ASFV113ASFV163 individual antigensASFV127ASFV105 Proliferation Assays:ASFV145 from immunised pigs withASFV154 Stimulation of lymphocytesASFV083ASFV006 Pool of 12ASFV194 3H-TdR uptake, cpmASFV132 0 100 150 200 250 300 350 400 450 50ASFV205ASFV170 ASFV002 PHA medium ASFV004 ASFV006 ASFV011 ASFV012 ASFV037 ASFV052 ASFV053 ASFV054 ASFV068 ASFV070 3H-TdR uptake, cpm ASFV07… ASFV07… 0 100 200 300 400 500 600 Pool of 22 ASFV083t ASFV105 ASFV111 ASFV122 ASFV128 2 antigens ASFV167 ASFV179 ASFV113 ASFV127 PHA medium Immune responses in pigs immunised with pools of antigens: Antigen pool complexity does not reduce T cell response level
  • Antigen pool complexity does not reduce antibody response Group 1 (pool of 22) vs. VP30 Group 3 (pool of 12) vs. VP301.6 127 pre 2 127 pre1.4 127 post 1.8 1.6 127 post1.2 1.4 1 1.20.8 1 0.80.6 0.60.4 0.40.2 0.2 0 0 394 395 410 419 420 393 396 398 404 405 406 Pig # Pig # Group 2 (pool of 22) vs. VP30 Group 4 (pool of 2) vs. VP301.6 127 pre 2 127 pre1.4 1.8 127 post 1.6 127 post1.2 1.4 1 1.20.8 1 0.80.6 0.60.4 0.40.2 0.2 0 0 397 407 409 411 412 424 403 417 418 421 423 Pig # Pig # ELISA assays
  • Summary of Progress: genome wide antigen screen• DNA vaccine and protein expression libraries complete• rVV library 47 complete• Immunome screening in pigs – conditions optimised and 47 antigens tested by DNA prime rVV boost• T cell and antibody assays used to rank ORFs for immune responses• Challenge experiments in progress
  • Future Priorities Vaccines• Attenuated vaccines: Rational strategy for attenuation• Better knowledge of cross-protection between genotypes- antigens involved in cross- potection• Optimised cell culture• Subunit vaccines: Identification of protective antigens especially those which induce CD8+ T cell responses• Incorporation and testing in host-restricted gene delivery systems
  • Future work vaccines• Subunit vaccines – complete screen for protective antigens• Test in pools in challenge experiments• Select best antigens and clone in host- restricted vaccine delivery vector
  • Acknowledgements IAH UK Biodesign Institute Arizona• Linda Dixon• Dave Chapman State University• Lynnette Goatley Center for Infectious Diseases• Fuquan Zhang • Bert Jacobs• Charles Abrams • James Jankovich• Emma Fishbourne • Greg Golden• Pam Lithgow Center for Innovations in Medicine• Derah Arav • Kathy Sykes • Mark Robida• Geraldine Taylor ANSES Ploufragan, France• Haru Takamatsu • Marie-Frederique le Potier• Katherine King• Chris Netherton • Evelyne Hutot• Josie Golding • Roland Carriolet• Pippa Hawes Univ. Victoria, Canada • Chris Upton www.virology.ca• Don King• Chris Oura• Carrie Batten• Geoff Hutchings
  • Genotypes of ASFV isolates Penrith et al., 2007Data from partial sequence of geneencoding p72 capsid protein
  • Antibody response following DNA prime rVV boost compared to infection Uninfected and ASFV-infected pigs vs. VP30 Pre/Post Immunization vs. 1.2 VP302.5 1 pre VP30 2 post VP30 0.81.5 0.6 1 0.40.5 0.2 0 0 1:100 1:500 1:2500 cont 01 cont 04 60 76 105 184 Uninfected and ASFV-infected pigs vs. VP72 Pre/Post Immunization vs. VP720.6 1.20.5 1 pre VP720.4 0.8 post VP720.3 0.60.2 0.40.1 0.2 0 0 cont 01 cont 04 60 76 105 184 1:100 1:500 1:2500