Better lives through livestock
Gene Editing in Veterinary Vaccine Development
- status of the science
Lucilla Steinaa
Principal Scientist
International Livestock Research Institute
CGIAR Webinar series: “Genome Editing in Agriculture:
Innovations for Sustainable Production and Food Systems”
6 October 2020

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Veterinary Vaccine Types
Attenuated vaccines
Live
Modified Live
Inactivated vaccines
Whole organisms
Recombinant vaccines
Subunit vaccines
Chimeric vaccines
Toxoid vaccines
Detox of toxin

3
Genetically Modified Vaccine Types
Vector-based. Inserted antigens for subunit vaccination
• Viral: Poxvirus, adenoviruses and herpes virus
• Bacterial: Salmonella, Listeria, Shigella (research)
Modified Live attenuated vaccines (viruses, bacteria, protozoa)
Using the modified organism as vaccine directly by deleting or
slowing expression of virulence factors
• Bacteria: Streptococcus, Escherichia, Salmonella
• Virus: Rabies, ILTV, PRRS, BVD

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Evolution of the GM Vaccine Types
Modification of
bacteria
First recombinantly
produced vaccine
Viral / bacterial
vectored vaccines
Attenuated GM
organisms
Human Hepatitis B
vaccine
Canarypox
vectored veterinary
vaccines
Poultry: Multivalent
Modified Live attenuated
Vaccines
Salmonella
vaccines
Multi-pathogen
vaccines

5Veterinary GM Vaccines (Vectored) – and Status Tawira et al. 2020

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Gene Edited Vaccines (Vectored) on Market (Source: Company Homepages)
Vaccine Company Molecular details Indication Development status
Raboral V-RG Merial
LAV. Oral Live vaccinia virus expressing Rabies
antigen
Rabies
Innovax®-ND-IBD MSD Animal
Health
LAV. Recombinant Turkey Herpesvirus
(Marek’s), with antigens from Newcastle
Disease Virus and Infectious bursal disease
Infectious Bursal
Disease, Marek’s
disease, Infectious
bursal disease
Marketed
VAXXITEK® HVT+
IBD+ND
Boeringer
Ingelheim
LAV. Recombinant Turkey Herpesvirus
(Marek’s) with antigens from Newcastle
Disese Virus and Infectious bursal disease
virus
Marek’s disease,
Infectious bursal
disease, Newcastle
Disease
Marketed
VAXXITEK®
HVT+IBD
Boeringer
Ingelheim
LAV. Recombinant Turkey Herpesvirus
(Marek’s) with antigens from Newcastle
Disease Virus and Infectious bursal disease
virus
Marek’s disease,
Infectious bursal
disease.
Marketed
Suvaxyn®CSF
Marker
Zoites
LAV. CP7_E2alfs. The main immunogen of CSF
virus “Alfort/187”, glycoprotein E2, in a
bovine viral diarrhea virus type 1 backbone
Classical swine fever Marketed

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Modified Live Vaccines on Market (European Medicines Agency)
Vaccine Company Molecular details Indication Development status
Hiprabovis IBR
Marker Live
Hipra Tk and gE deleted virus
Infectious Bovine
Rhinotracheitis
Bovine
Licensed Europe
2011
Equils StrepE Intervet
Live streptococcus strain, aroA deletion
Incapable of making folate
Respiratory disease
Horses
Licensed Europe
2004
Poulvac
Ecoli
Salmonella
Zoetis
aroA deletion
Incapable of making folate
Colibacillosis
Chicken, turkeys
Salmonellosis
Chicken
Licensed Europe
2012
Bovela
BVDV
Boeringer
Ingelheim
Deletion of genes in type 1 and 2
Bovine Viral Diarrhea
Cattle
Licensed Europe
2014
Suvaxyn-PRRS-
MLV
Zoetis Modified
Abortions, still birth
due to PRRS virus
Swine
Licensed Europe
2017

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Genetically Modified Vaccine Types
Ramezanpour et al. Vaccine. 2016

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GM Vaccines for Humans
• The first: IMO-JEV, Japanese Encephalitis vaccine based on
yellow fever vector. 2010 Japan.
• The second: Dengvaxia, Japanese encephalitis vaccine, two
genes from JEV inserted in yellow fever backbone. 2015, many
countries, Europe, USA

10Gene Edited on More Complex Pathogens
Vaccine Molecular details Indication
Development
status
Referece
Protozoa
(Apicomplexan)
Plasmodium falciparum (humans)
Liver-stage antigens has been targeted
Timed genome editing
Gene editing for increased immunity
Self destructive parasites
Malaria Research
M.C. Lee, et al.
Microbiol Mol Biol Rev
2014
Protozoa
Trypanosoma cruzi (humans)
Deletion of genes for flagellum attachment (cruzei)
Chagas disese Research
Reviewed by
Yoagubat et al. Trends
in Parasitol 2020
Protozoa Trypanosoma brucei (humans) Sleeping sickness Research
Reviewed by
Yoagubat et al. Trends
in Parasitol 2020
Protozoa Trypanosoma vivax (animals) Trypanosomiasis Research
D ’Archivo et al. Plos
Negl Trop Dis. 2011
Protozoa
(Apicomplexan)
Toxoplasma gondii (human and animals) Toxoplasmosis
Research Shen et al. Methods
Mol Biol. 2017
Protozoa
(Apicomplexan)
Crytosporidium spp (human and animals) Cryptosporidiosis
Research
Beverley. Nature 2015
Protozoa Babesia bovis (animals) Babesiosis
Research Asada et al. Mol
Biochem Parasitol
2012

11Gene Editing of Pathogens at ILRI
for Vaccines and Research
African Swine Fever (ASF)
• Caused by a large DNA virus
• Present in about 26 countries in Africa – constraint
to the pig farming
• Major problem in Eastern Europe, Russia and Asia.
Contagious Bovine Pleuropneumonia (CBPP)
• Caused by a Mycoplasma
• One of the most serious cattle diseases in Africa.
• Imposes an estimated minimal cost of > 100 M €/year
in Africa and restricted trade.
Technologies:
CRISPR-Cas editing
Synthetic biology
Genome transplantation

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CRISPR Modification of African Swine Fever
Virus backbone: genotype IX isolate from outbreak in Kenya at the
Ugandan border Onzere et al., Virus Genes. 2018
10 different viruses produced, ready for test in animals

Modify fragments of ASFV
Assemble genomes in yeast
Reconstitute live virus
Test infectivity and growth in
tissue culture and in vivo
Rapid Modification of ASFV to Obtain Vaccine Candidates
Design
Build
Test
Transform into yeast
Isolate plasmid
Propagate in coli
Cut out and ligate loops loopsTransform
YCpBac
1 2 3 1 2 3 1 2 3 wt wt wt
F1R1 F2R2 F1R2
F1R1
F2R2
F1R2

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Mmc – genome transplantation – model
Schieck et al. Mol Microbiol 2016, Jores et al. JID 2018
GM12 YCPMmyc1.1 Δglf
GM12 YCPMmyc1.1
Attenuation of Mycoplasma via Knock-out of Genes Involved in
Polysaccharide Biosynthesis

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What Is the Next?
• Refinement of technologies
- Inducible systems, self-destruction
• DNA plasmid-coded attenuated viruses
- Optimizing delivery
• Bacterial vectors: Lots of ongoing clinical
trials
• Multivalent vaccines:
Many pathogens in one
• More complex pathogens:
Helmiths

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Mycoplasma work
Elise Schieck (ILRI)
Jorg Jores (ILRI)
Carole Lartigue (CIRAD)
Sanjay Vashee (JCVI)
African swine fever work
Hussein Abkallo (ILRI)
Nicholas Svitek (ILRI)
Elias Awino (ILRI)
Bernard Odour (ILRI)
Lucilla Steinaa (ILRI)
Sanjay Vashee (JCVI)
Walter Fuchs (FLI)
Acknowledgements

THANK YOU