The Role of CD163 and SIGLEC1 in Sensitivity to

Porcine Reproductive and Respiratory

Syndrome Virus Infection

Genetic Engineering of Pigs at Missouri• Cancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1)• Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CAT+)• Cardiac Development (Floxed FGF8)• Cystic Fibrosis (CFTR-/-; CFTRΔF508/ΔF508; CFTRΔF508/ΔF508, Tet-on CFTR+; CFTRΔF508/ΔF508, FABP2-CFTR+; shRNA CFTR)• Diabetes (ssGIP-hINS+)• Disease Resistance (SIGLEC1-/-; CD163-/-; CD163-/Δdomain 5; CD163-/Domain 5 swap with domain 8 of hCD163L; CD163-/-,

SIGLEC1-/-; CD1D-/-)• Enhancing Meat Quality (hFAT1+)• Muscular Dystrophy (DMD+/Δexon 46)• Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+)• Regenerative Medicine (IL2RG+/-; RAG2-/-)• Retinitis Pigmentosa (RHO P23H)• 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-/-)

• Plus others that I can’t talk about- over 45 different modifications and over 1,000 cloned pigs.

Outline• Model of Infection for PRRSv• SIGLEC1• CD163• Timelines & From Here to Where?

• 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 the Sialoadhesin Gene and Targeting Vector Design.

Southern Probe

Prather et al ‘13 J Virology

• A targeted clone (4-18) was identified and used both for somatic cell nuclear transfer and to propagate for Southern blot analysis.

• Screened >2,000 colonies: efficiency ~7.0 x 106

Transfection

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

Clinical Outcome• Assessed daily for clinical signs

– Pigs co-housed – No apparent clinical signs before infection– Day 3 after infection all pigs began to exhibit mild respiratory signs (sneezing,

coughing, increased breathing)– These resolved within 1- to 2-weeks. In a single WT pig more severe signs

reappeared on day 28.• Euthanized at the end of the study

– Histology- Lungs- no difference as all had moderate to severe multifocal interstitial pneumonia.

– Collected alveolar macrophages.

Prather et al ‘13 J Virology

Do the SIGLEC1-/- pigs express SIGLEC1?

• Flow Cytometry on porcine alveolar macrophages at Kansas State University- Bob Rowland.

CD163SIGLEC1Wild-Type pig

2nd Ab

Prather et al ‘13 J Virology

CD163SIGLEC1Wild-Type pig

2nd Ab

Do the SIGLEC1-/- pigs express SIGLEC1 in PAMs?

SIGLEC1-/- pig

CD163SIGLEC12nd Ab

Prather et al ‘13 J Virology

Prather et al ‘13 J Virology

SIGLEC1

• Virus in blood that killed cells in vitro- cytopathic effect.

Prather et al ‘13 J Virology

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

Timeline• Conceive Idea – June ‘02.• Search for $$$• Founder male heterozygotes – Born Jan ‘11.• Founders reach puberty Oct-Sept ’11.• F1’s born (heterozygous males and females) Nov ’11-Jan ‘12.• F1’s reach puberty summer ‘12.• Homozygous Knockout animals born Oct ‘12.• Challenge experiments began Nov ‘12.• Publication ’13 almost 11 years later.

Prather et al ‘13 J Virology

SIGLEC1 Conclusions• SIGLEC1-/- is not embryonic lethal.• Potential problem- KO mice have subtle changes in T- and B-

cell populations, and reduced immunoglobulin M (Oetke et al ‘06).

• SIGLEC1-/- may have altered IgG response, but variation was high and sample size was small.

• SIGLEC1-/- did not affect susceptibility to PRRSv.• Could cells from these pigs be useful diagnostic tools?

Prather et al ‘13 J Virology

The Case for CD163• A series of experiments similar to that for SIGLEC1

provided evidence that CD163 is not only an entry mediator but also facilitates infection (as outline by Welsh & Calvert ’10). Other candidates include CD151, VIM and CD209.

CD163 CD163 is a member of the scavenger

receptor cysteine-rich (SRCR) superfamily

17 exons, 16 introns

• Determine which extracellular domains were required for PRRSV infectivity

• HEK293T were transfected with SIGLEC1 and CD163 deletion constructs

• Cells were inoculated with PRRSv for 24 hr and fixed

• Number of infected cells were counted

Deletion Constructs

Van Gorp et al 2010 J Vir

Chimeric Mutants• CD163 is a member of gene family

– hCD163L (CD163-like)– 12 extracellular domains– Does not function in infectivity

• Swap domains from hCD163-like and CD163 and determine infectivity

Van Gorp et al 2010 J Vir

Human CD163

Human CD163L

Two Proposed Targeting Approaches• Traditional knockout by homologous recombination and

addition of a stop codon• Domain Swap

– Remove extracellular domain SRCR5 from CD163– Replace with extracellular domain 8 from hCD163L

• Haptoglobin-Hemaglobin – SRCR #3• Erythroblast – SRCR #2• TNFSF12 (aka TWEAK) – SRCR #1-4 & #6-9 • Bacteria – SRCR #2• PRRSv- SRCR #5

Advantages of a Domain Swap

But is a Domain Swap Necessary?• Haptoglobin’s ability to promote binding of hemoglobin to

CD163 in human has high affinity, but not in mice.• In mice CD163 accounts for a small part of hemoglobin

clearance.• Knockout of CD163 results in “No apparent phenotype

change… and offspring were viable and fertile”. Etzerodt et al ‘12. Antioxidants & Redox Signaling, DOI: 10.1089/ars.2012.4605

CRISPR-Mediated 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.

CD163 Timeline (SCNT)• Genomic Sequence Confirmed and CRISPRs

designed (1 day, if good EST data available)– 7-18-13

• CRISPRs pairs ordered from IDT (1 day)– 7-19-13

• CRISPRs pairs annealed and ligated and transformed

• Bacterial Colonies picked, propagated and sequence confirmed

• Positive colonies re-propagated in larger preps for transfections

– 7/22/13-7/26/13 (1 week)• Design Assays for smaller and large deletions

– This project had large assays– Small assay 435 bp was developed (2

days)

• Cells ready for transfection 8-7-14• First Transfection with CRISPRs (14 days)

– 8/9/13-8/23/13• Colonies picked and genotyped (2 days)

– 8/23/14-8/24/14• Positive Colonies were propagated and

sequence confirmed – 8-26-13 (this took along time)

• Nuclear Transfer – 9/19/13

• Embryo Transfer– 9/20/13

• First Pigs modified by CRISPRS 1/13/14

Total Time by SCNT: 5.5 - 6 months

Timelines• Engineering Somatic Cells (1 month to 2 years)• SCNT/ Zygote injection (1 week)• Gestation (3 months, 3 weeks & 3 days)• Puberty (6-9 months)

Weaned wildtype and CD163 edited piglets prior

to transport to Kansas State University.

Patent Pending

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

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

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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?

Clinical Signs

Whitworth et al.

• Surface expression of CD163 or CD169 on porcine alveolar macrophages (PAMs) was measured using flow cytometry

• PRRSV viremia in serum measured using commercial Tetracore assay with standards. Results reported as log10 templates per PCR reaction

• PRRSV N protein specific antibody response measured by Luminex. Results reported as S/P ratio

S/P = (Sample-BKG)/(Positive-BKG)• Histopathology and scoring of microscopic lesions

performed by a board certified anatomic pathologist, Dr. Giselle Cino

Methods

Whitworth et al.

Viremia and Antibody

Whitworth et al.

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.

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

Expression of CD163 and CD169 (SIGLEC1) on PAMs

Whitworth et al

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

Van Breedam et al., 2010

CD163 is a Gatekeeper!

From Here to Where?

• Answer Basic and Applied Questions about Human Medicine and Domestic Animal Biology

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

Final Thoughts• 10th International Conference on Pig Reproduction

June 11-14, 2017, University of Missouri, Columbia, MO

• Open Postdoc Position: Molecular Biologist that wants to create additional models. PratherR@Missouri.Edu