dr. randall prather - prrs resistant pigs

The Role of CD163 in the Sensitivity to the Porcine

Reproductive and Respiratory Syndrome Virus

Outline• Genetic Engineering • Porcine Reproductive &

Respiratory Syndrome Virus–Traditional Genetic Engineering

• SIGLEC1–CRISPRs

• CD163

Swine are being used extensively as biomedical models of human health

– Bioengineering– Cancer– Cardiovascular Disease/Atherosclerosis– Cognitive Behavior– Cutaneous Pharmacology/Wound Repair/Dermatology – Diabetes– Immunology– Lipoprotein Metabolism– Nutrition– Organ Transplantation– Orthopedics– Ophthalmology – Physiology– Regenerative Medicine

Adapted from Prather et al 2013

Genetic Engineering(CRISPRs, Zinc Fingers, TALENS, Homologous Recombination)

Transgene InjectionNuclease Injection

Oocyte TransductionSperm-Mediated Gene Transfer

Design & Build Constructs or

Guides

Genetic Engineering of Pigs at Missouri

• Cancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1)• Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CAT+)• Cystic Fibrosis (CFTR-/-; CFTRΔF508/ΔF508; CFTRΔF508/ΔF508, Tet-on CFTR+;

CFTRΔF508/ΔF508, FABP2-CFTR+; shRNA CFTR; MUC5AC-/-,MUC5B-/- )• Diabetes (ssGIP-hINS+)• Disease Resistance (SIGLEC1-/-; CD163-/-; CD163-/Δdomain 5; CD163-/-, SIGLEC1-/-;

TMPRSS2-/-; CD1D-/-)• Enhancing Meat Quality (hFAT1+)• Muscular Dystrophy (DMD+/Δexon 46)• Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+)• Regenerative Medicine (IL2RG+/-; RAG2-/-)• Retinitis Pigmentosa (P23H RHO +)• Spinal Muscular Atrophy (SMN+/-; SMN+/-, hSMN2+)• Suicide Prodrug for Liver Transplantation (ALB-TK1+; AFP-CDA+) • Tracking/Tools (CAG-eGFP+; CMV-eGFP+; ZP3-CRE; UBC-Tomato+; CAG-

Tomato+; UBC-nls eGFP+; PSMA1-eGFP fusion protein +)• Xenotransplantation (GGTA1-/-; GGTA1-/-, CD55+; GGTA1CD55/+; GGTA1-/-,

CD55+, CD59+, ENTPD1+, THBD+; SIGLEC1-/-)

• The PRRS virus (PRRSv) was first detected in the U.S. in 1987 (Keffaber et al ‘89) and in Europe in 1990 (Wensvoort et al ’91)

• PRRSv replicates in macrophages– induce prolonged viremia and can cause persistent

infections that last for months• It also predisposes infected pigs to other

bacterial and viral pathogens

Porcine Reproductive and Respiratory Syndrome (PRRS)

Animal Wellbeing Cost• Vaccinations to date have been ineffective

– Non- neutralizing antibodies actually enhance viral replication in alveolar macrophages

– Antibody dependent enhancement (ADE)• Sows/Gilts

– severe reproductive failure and a high rate of late abortion – Early farrowing– Decreased litter size, Increased number of mummies

• Boars– Low libido – Fever, low sperm count

• Young and growing pigs– Pneumonia, Diarrhea, Mortality of 12-15%

http://www.thepigsite.com/diseaseinfo/97/porcine-reproductive-respiratory-syndrome-prrs

Economic Cost• Costs $660,000,000 annually in North

America (Holtkamp et al ‘13)

• Costs €1,500,000,000 annually in Europe (European PRRSpecive ‘15)

• Translates to ~$6,000,000 each day!• Doesn’t include Asia.

Other Costs• Sustainability costs• Threat to food security• Psychological & emotional costs

PRRSv Infectivity Was Thought to Result from 3 Specific Entry Mediators

– Initial binding with heparan sulfate– Binding/Internalization by Sialoadhesin– Internalization/uncoating of the virus by CD163

Van Breedam et al., 2010

Heparan Sulphate Binding

Binding and Internalization by Sialoadhesin(SIGLEC1)

Uncoating of the virus by CD163

Genome Release in endosomes dependent on low pH

Van Breedam et al., 2010

Organization of the Sialoadhesin Gene and Targeting Vector Design

Southern Probe

Prather et al ‘13 J Virology

Enucleation

Fusion or Injection and Activation

Development

Transfect or Transduce and Select

Synchronize?Nuclear TransferUS Patent #6,211,429

SIGLEC1+/- pigsBorn 1/16-18/2011 Patent Pending

Prather et al ‘13 J Virology

First SIGLEC1 -/- Pigs (F2 pigs) BornOctober 18, 2012

4 SIGLEC1 -/-

4 SIGLEC1 +/-

4 SIGLEC1 +/+ Wild Type

Prather et al ‘13 J Virology

PRRSv Challenge• Piglets were shipped to Kansas State

University• After 1 week acclimation

– Low passage PRSSV isolate (KS-06: North American isolate collected in 2006)

– 105 TCID50 of virus diluted in 3 mL• ½ intramuscularly• ½ intranasally

– Blood sampled on days 0, 4, 7, 14, 21, 28 & 35.

Prather et al ‘13 J Virology

• Viral RNA/nuclei acidPrather et al ‘13 J Virology

PRRSV RT-PCR

SIGLEC1 Conclusions• SIGLEC1-/- is not embryonic lethal.• SIGLEC1-/- did not affect susceptibility

to PRRSv.

Prather et al ‘13 J Virology

CRISPRsClustered Regularly Interspaced Short Palindromic Repeats

• Bacterical immune system that degrades foreign genetic elements (plasmids, phages).

• Can edit the genome at a very high efficiency.

• Uses two main components: a guide sequence and a Cas9 protein.

Zygote Injection

Gene Editing• Non-Homologous End Joining• Can result in changing a handful of

base pairs• Thus can remove exons/domains or

knockout a gene.• Swine genome is 2,700,000,000+ base

pairs.

Weaned wildtype and CD163 edited piglets prior to transport

to Kansas State University.

Patent Pending

Whitworth et al, ‘15Nature Biotechnology

Pigs Included in the Study

• 3 piglets predicted to be null (CD163-/- )• 8 WT piglets

– One WT piglet humanely euthanized on day 1, due to poor body condition and not included in the study

• K-State veterinarians and staff blinded to genotypesPiglet ID Predicted

TranslationMaternal Allele Paternal Allele

#43 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron

#55 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron

#40 CD163-/- 7 bp addition in exon 7 11 bp deletion in exon 7

Whitworth et al, ‘15Nature Biotechnology

Experimental Design• Identity blinded to the crew at KSU• Housed in the K-State BL-2 LARC facility• Allowed to acclimate for 3 days after arrival at facility• Challenge IM and IN with 105 TCID50 of NVSL 97-7985.

(Standard lab isolate from 1997, relatively “hot” in terms of replication and pathogenesis)

• Pigs were maintained in the same pen; therefore, all pigs constantly exposed to virus

• Monitored daily for clinical signs• Blood collected on days 0,4,7,11,14,21,28,35 and weights

collected at least weekly• Study terminated 35 days after infection• Lung lavage for PAMs• Lungs and tissues removed for histopathology

Whitworth et al, ‘15Nature Biotechnology

From the Beach in Florida!

http://www.scuba-diving-smiles.com/palm-tree-clipart.html

Whitworth et al, ‘15Nature Biotechnology

Clinical Signs• Fever was considered positive if it was ≥ 104°F• Respiratory scores ranged from:

– 0: Normal, to – 1: mild dyspnea and/or tachypnea when stressed (when

handled), – 2: mild dyspnea and/or tachypnea when at rest, – 3: moderate dyspnea and/or tachypnea when stressed (when

handled), – 4: moderate dyspnea and/or tachypnea when at rest, – 5: severe dyspnea and/or tachypnea when stressed (when

handled), – 6: severe dyspnea and/or tachypnea when at rest. Is there

evidence of diarrhea (grade) or vomiting?

Whitworth et al, ‘15Nature Biotechnology

Clinical Signs

Whitworth et al, ‘15Nature Biotechnology

Data provided by Maureen Kerrigan

Typical Viremia Response

Over 3000 pigs challenged at Kansas State with a response like these.

Viremia

Whitworth et al, ‘15Nature Biotechnology

Day After Infection

Antibody

Whitworth et al, ‘15Nature Biotechnology

Day After Infection

Lung Pathology

• CD163+/+ pigs had interstitial edema with the infiltration of mononuclear cells and the mononuclear infiltrate consisted mainly of lymphocytes and plasma cells and lesser numbers of macrophages. In contrast there was no evidence for pulmonary changes in the CD163-/- pigs.

Whitworth et al, ‘15Nature Biotechnology

Pig Genotype Description Score*

41 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers of lymphocytes and macrophages.

3

42 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers of lymphocytes and macrophages.

3

47 Wild Type 75% multifocal infiltration with of mononuclear cells and mild edema. 2

50 Wild Type 75% moderate infiltration of mononuclear cells within alveolar spaces and around small blood vessels. Perivascular edema.

3

51 Wild Type 25% atelectasis with moderate infiltration of mononuclear cells. 1

52 Wild Type 10% of alveolar spaces collapsed with infiltration of small numbers of mononuclear cells.

1

56 Wild Type 100% diffuse moderate interstitial infiltration of mononuclear cells. Interalveolar septae moderately thickened by hemorrhage and edema.

4

40 CD163-/- No changes 043 CD163-/- No changes 055 CD163-/- No changes 0*Lung lesion scores calculated according to Halbur et al. 8 Vet Pathol. 1995 Nov;32(6):648-60

Lung Pathology

Whitworth et al, ‘15Nature Biotechnology

Overall- Conclusions

• SIGLEC1-/- pigs are not resistant to PRRSv.

• CD163 -/- pigs are resistant to PRRSv.• Working on challenging other

genotypes.• Working on challenging with other

isolates (multiple Type 1 and Type 2).

Heparan Sulphate Binding

Binding and Internalization by Sialoadhesin

Uncoating of the virus by CD163

Genome Release in endosomes dependent on low pH

CD163 is a Gatekeeper!

Van Breedam et al., 2010

From Here to Where?• Technology Permits Answering Basic

and Applied Questions about Human Medicine and Domestic Animal Biology

• Any Genetic Modification that you can imagine, we can probably build.– Miniature Pig– Domestic Pig

• CD163-/- Entry Into Production– Growth and Performance

Zygote InjectionsKiho LeeLee Spate

CRISPR and Targeting Vector DesignKevin WellsKristin Whitworth

Transfection and GenotypingMykel AndersonMariah ThomasJoshua Benne

Nuclear Transfer and Embryo TransferJoshua BenneStephanie MurphyJennifer TesonJiude MaoClifton Murphy

Surrogate and Piglet CareMelissa SamuelJason DowellTricia MeyerEntire Prather Lab

Funding SourcesGenus plcThe Christopher Columbus Fellowship FoundationFood for the 21st CenturyUSDA ARS

Genus plcAlan MilehamDave McLarenJon Lightner

SIGLEC1-/-

ProjectJon GreenTina Egen

Kansas State UniversityBob RowlandBenjamin TribleMaureen KerriganCatherine EwenAda Cino-OzunaBhupinder Bawa