US Veterinary Immune Reagent Network

Project DirectorsCynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Erin Bromage, University of Massachusetts Dartmouth

John Hansen, WFRC-USGS-Biological Resources Division Joanna LaBresh, Kingfisher Biotech Hyun Lillehoj, USDA-ARS Beltsville Joan Lunney, USDA-ARS Beltsville

Bettina Wagner, Cornell University Melanie Wilson, University of Mississippi Medical Center

www.vetimm.org

Funded by:USDA-CREES 2006-2009;USDA-NIFA 2010-2014

ObjectiveA major obstacle to advances in veterinary immunology and

disease control is the lack of sufficient immunological reagents specific for ruminants, swine, poultry, equine and aquaculture species.

The goals of the project are to develop sets of reagents to begin to address this dearth.

Targets of US-VIRN• Cytokines and chemokines:

bioactive recombinant proteins as well as Abs to them

• Ab to cytokines & chemokines: mAb pairs for ELISA, multiplex and ELISpot assays (in some cases this may include polyclonal Abs); single mAb for intracellular staining for cytokines

• mAbs to Igs isotypes and IgG subclasses

• mAb anti-CD molecules and T cell receptors: mAb reactive with native molecules and used to stain viable cells for flow cytometric analyses including cell sorting to purify cells by flow cytometry or magnetic beads; for anti-cytokine receptors mAb that block function and signaling

Kingfisher Biotech

Presented by Joanna LaBresh

Kingfisher Biotech

14 SpeciesBovine, Canine, Catfish, Chicken, Dolphin, Equine, Feline, Guinea Pig, Mouse, Ovine, Rabbit, Rat, Swine, TurkeyProduct TypesProteins (>175), Antibodies(>140), ELISA Pairs, (>50) ELISA Sets (25)

Proteins

• Expressed in yeast because of the advantages of yeast– Expressed into media– Properly folded– Post-translationally modified– No endotoxin

• We never use tags

Proteins expressed in yeast

Note: not all targets in list were requested for all

species

Those check-marked in Red have ELISAs developed by

KingfisherBiotech called VetSets

Cytokine/ chemokine Cattle Swine Horse PoultryIL-1β & α √ √ turkey

IL-2 √ √ √ IL-4 √ √ √ √ IL-5 √ √ IL-6 √ √ √ √ IL-8 √ √ √ turkey

IL-10 √ √ √ IL-13 √ √ √ In progressIL-15 √ √ √ IL-16 √

IL-17A √ √ √IL-17F In

progress √ √IL-18 √ √ IL-21 √ √IL-22 √ √ IFNα √ √ √IFNβ √ √ √ IFNγ √ √ √ √ CCL2 √ √ √ CCL3 √ √ CCL4 √ √ √ CCL5 √ √ √

CCL20 √ CCL11 & CXCL9 √ √

CXCL10 √ √ √ CXCL11 √ √ TNFα √ √ √ √ (TNF-SF)

GM-CSF √ √

Progress US-VIRN target

Example: ELISA kit results

New Products

• Our pipeline is dynamic– We receive a couple of requests a week. – We are happy to hear what products our

customers would like to further their research

Catfish

Presented by Mel Wilson

For catfish, the highest priority reagents have been and are mAbs that recognize B and T cell subsets and macrophages.

Antibodies to inflammatory cytokines have also been requested.

Our goal is to prove specificity by FACS, western blotting, and immunoprecipitation with peptide sequencing.

Peer review publications: 8Abstracts: 16Oral presentations:17

VIRN:Catfish Species Report

Catfish Progress & Planning

Anti-IgD (IgG1k) and Anti-IgL s (IgMk) mAb

Available mAbsAnti-IgM Anti-IgDAnti-IgL k F Anti-IgL sAnti-IgL k G

B cell subpopulationsIgM+/IgD+

IgM+

IgD+

Anti-TCR alpha (IgG1k) mAb

T cell line TS32.15 PBL CRP 14

19 % 20 %

PBL CRP 16

Anti-TCR alpha Anti-TCR alpha Anti-TCR alpha

TroutPresented by Erin Bromage

IgT

CD79α

CD8

TCRβ

CD4rel

CD4

CD80

CD83

IgD

CD3e

LCK

MoleculeFull length

cDNAProtein

producedMice

ImmunizedPolyclonal sera

reactivity mAb FusionmAb

reactivityAssay

development

Current Position (April 2011) Years 3-4 Seeking alternative external funding or collaborations

IL1B-2

CXCR4

Trout Progress & Planning

IgL3

IgL2

IgL1

secIgM

TCRβ

C3d

IL6

IgL4

Anti –IgD mAbs

Anti-CD3e mAbs

Anti-IL1B-2 mAb

Chicken

Molecule Full length cDNA

Protein produced

Mice Immunized

Polyclonal sera reactivity

mAb Fusion

mAb reactivity

Assay development

IL-2IL-1β

IL-6IL-7

IL-10IL-12p35IL-12p40

IL-15IL-16

IL-17FIL-17A

IL-18IL-21IL-22

CD80CD25

CD83CD86

IL-13

Current Position

At the end of Mar 2013

At the end of April 2014

IL-4

IL-8

Chicken Progress & Planning

Chicken Progress & Planning

CCL20CCL4

CXCL14GMCSF

IFN-γLITAF

LTMIF

TL1A

IL-10R-βIL17RIL21R

IL1R1

IL-7R

VEGFΒ-defensin8

Molecule Full length cDNA

Protein produced

Mice Immunized

Polyclonal sera reactivity

mAb Fusion

mAb reactivity

Assay development

IL-5R

NK lysin

IFN-α

TGFB4

Current Position

At the end of Mar 2013

At the end of April 2014

Seeking external funding

Reagents Completed through to mAbs

• 11 mAbs to chicken protein - IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γ

• Test methodWestern: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γ

Flow cytometry: CD25, CD80, CD83, CD86

Tissue staining: CD25, CD80, CD83

Neutralizing functional assay: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, IFN-γ

23

148

98

64

50

IL-1β

14898

64

50

36

22

• Western IL-8

14898

64

50

36

22

IL-6

IFN-γ

mAb #1 #2

IL-18( kDa)

148

98

64

50

36

16

• Flow cytometry

CD25 CD83

C

A B

Flow cytometric analysis of mAbs to chCD25 (A), CD80 (B) and CD83 (C) Using CHO-chicken cells

Tissue staining with mAbs to CD25, CD80 & CD83

Immunolocalization of chCD25 in tissues. Bursa of Fabricius (A), spleen (B), and intestinal duodenum (C)

Expression of chCD80 on LPS-stimulated dendritic cells.

Immunolocalization of chCD83 in lymphoid organs. (A) Caecal tonsil, (B) bursa of Fabricius, and (C and D) spleen

Neutralization of IL-18 bioactivity by chIL18 mAbs.

Inhibition by mAb to CD25 on IL-2-dependent splenocytes proliferation.

Effect of anti-chCD80 mAb on IL-2 driven lymphoblast cell proliferation.

Functional assays with mAbs (anti-IL-18, CD25, CD80 & IL-1)

26

MediaCon A 500

125 31 8 2 8 320

0.2

0.4

0.6

0.8

Opt

ical

den

sity

(450

nm

)

IL 1β(ng/ml) mAb Dilution factor

Functional activity of chIL-1β on thymus lymphocyte proliferation and its mAb’s neutralization effects.

Horse

Used both yeast and mammalian-expressed cytokines & chemokines for immunization

COMPLETED:IL-2

IL-4 (mamm)IL-5IL-6

IL-10 (mamm)IL-17A

IFN- (mamm)CCL2CCL3CCL5

CCL11

GM-CSF

CXCL9

CCL11

CCL5CCL3CCL2

IL-15

IL-12

IL-8

IL-6

IL-13

IL-1

IL-5

IL-4IL-2

MoleculeFull length

cDNAProtein

producedMice

immunized mAb FusionmAb

reactivityAssay

development

Current Position (1/2012)

Continued during this funding period

CXCL10

IL-17A

Horse Progress & Planning: Cytokines & Chemokines

Expression vector/

Transfectant

IL-18TGF-

Mabs to yeast protein did not react with native cytokine

No mAbs with E. coli or yeast expressed proteins

Yeast protein development/ mAb development not planned

(antibodies exist)IFN-

IFN-

Completed:CD14CD23CD25CD16CD40

FcRITCR

Molecule Full length cDNA

Protein produced

Mice Immunized

mAb Fusion

mAb reactivityflow cytometry

Protein purified

Current Position (1/2012) Procedure repeated

CD16

CD23

Foxp3

IgD

CD28 CD25

FcRI

CD40

TLR2

IFNAR

TCR

TCRTCR

CD19

CD34

CD105

NCR2

NCR3

Continued

Horse Progress & Planning: Cell Surface Molecules

Done by collaborator lab

Characterized and published antibodies (available through Cornell):Anti-equine IL-4Anti-equine IL-10Anti-equine IFN-Anti-equine CD14Anti-equine CD23

Characterized antibodies (available soon):Anti-equine IL-2 Anti-equine CCL2 Anti-equine CCL3Anti-equine CCL5Anti-equine L-17AAnti-equine CD25Anti-equine CD16http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htmhttp://www.vetimm.org

Characterization ongoing: anti-IL-5, anti-CCL11

Equine cytokine multiplex assay (IL-4, IL-10, IL-17, IFN-, IFN-) available through the Animal Health Diagnostic Center at Cornell (ahdc.vet.cornell.edu)

Monoclonal antibodies to equine cytokines, chemokines and cell surface molecules developed at Cornell

Anti-equine CD14 mAbs

(Kabithe et al. 2010, Vet Immunol Immunopathol)

Magnetic cell sorting, CD14 mAb 105

CD14 mAb 59 (lane 2)

Mammalian expressed equine CD14 was used for immunization

Anti-equine CD25 mAb

PBMC, 48h, medium

CD4

CD2

5

PBMC, 48h, PWM

CD4

CD2

5

3 mAb clones, tested on PBMC of various horses in 2011, given to two equine labs outside US-VIRN for additional specificity testing who confirmed our results

Mammalian expressed equine CD25 was used for immunizations

Anti-equine IL-17A mAbs

Equine PBMC, non-stimulated or stimulated with PMA/ionomycin, stained and analyzed by flow cytometry

Anti-equine L-17A mAbs were made using yeast expressed IL-17A

Applications for anti-equine cytokine mAbs produced

Anti-IL-17A mAbs have been also tested now and work well for ELISA, Multiplex assay and FACS.

Swine

CCL2

Molecule Full length cDNA

Protein produced

Mice Immunized

mAb Fusion

mAb reactivity

Assay development

CCL3L1

Protein Purified

Protein Bioactivity

Current Position Future Year Plans

IFN1,6

CXCL10CXCL11

CCL4CCL5

CXCL9

IFN

IL-7

TNF

IL-15IL-13

IL-17A

IL-22IL-23A

IL-9

IgG3

2011-12 DHS/NIFA funding plans

IgG2IgG1

IL-17F

IL-6

CCL20CCL25CCL28

IFN

IgG5

Continuing

IL-21

Swine Immune Reagent Planning – Cytokine, Chemokines and Igs

CXCR3

Molecule Full length cDNA

Protein expressed

Mice Immunized

mAb Fusion

mAb reactivity

mAb Epitope defined

Renewal Future Year Plans

CXCR5

TCRTCR

IL-4RIL-13R

CD1c

NKp30

NKp46

IL-17R

NKp44Austria

Current Position (Jan. 2012] 2012 DHS funding plansVIRN Renewal 2012-13 Plans

CD19

IFN R

Swine Immune Reagent Planning – Cell Surface Molecules

Expanded Swine cytokine Fluorescent Microsphere Immunoassay (FMIA) (magnetic bead assays)

Lab developed FMIA* 6-plex detected IL-1b, IL-4, IL-8, IL-10, IL-12, IFNa; As a result of US Toolkit efforts** have recently added CCL2 to multiplex

* Lawson S, Lunney JK, et al. Vaccine 32: 5383-5391, 2010.

**Wagner, Lunney et al. in preparation

Increased levels of serum CCL2 were associated with pigs which had higher serum PRRS virus burden

Souza, Araujo, Lunney et al. in preparation

P

P<0.05 repeated measures combined with 0-21 dpi viral load [low vs high]

Cattle

IL-1β

IL-15

IL-12p35

IL-10

IL-8

IL-7

IFN-

IL-2

IL-5

IL-4

IFN-β

IFN-α

CXCL11

CXCL9

CCL5

CCL2

CXCL10

MoleculeFull length

cDNAProtein

producedMice

immunized mAb FusionmAb

reactivityAssay

development

Current Position Continuing With new funding

IL-17A

IL-6

IL-17F

Cattle Progress & Planning: Cytokines & ChemokinesExpression

vector/Transfectant

IL-12p40

IL-13

TNFα

Bioactivity determined

CCL11

IL-18

Commercially available

Cattle Progress & Planning: Cell Surface Molecules

IL-23R

MoleculeFull length

cDNAProtein

producedMice Immunized mAb

FusionmAb

reactivityProtein Purified

Current Position Continuing in current grant With new funding

IL-10R

TCRγ-C3

TCRδ–V1

TCR α

CCR7

TCRβ

CD107a

CD207

TCRδ-V2

TCRδ-V3

TCRδ-V4

TCRγ-C5

Monoclonal antibodies to bovine IL-17F using mammalian-expressed protein – 3 parent clones available

Monoclonal antibodies made to bovine IL-17A using mammalian-expressed protein:

Subclones of 6F2

6F2-1 6F2-2 6F2-4 6F2-7 6F2-8 6F2-9 6F2-10 6F2-110.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

HK Bt IL17A

KF Bt IL17A

HK Ss IL17A

HK Ss IFNb

BSA

Mammalian-expressed swine IL-17AMammalian-expressed bovine IL-17A

2C1 4B10 5E3 8G8 11C2 2G90

0.10.20.30.40.5

Sw IL-17A fusion screening 5/16/12

Sw IL-17ABo IL-17ABo IL-17FCCL3L1PBS

Clone

OD

405

Mouse titers to Yeast (KF)-expressed bovine CCL2

100 1000 10000 1000000

0.5

1

1.5

2

2.5

KF Bovine CCL2 Titer ELISA 11-13-12 Mouse #1

Bo CCL2 KFSw IL-17F KFSw CCL3L1 293tBo IL-6 293tBSAPBS

Serum dilution factor

OD

405

WHERE CAN I GET THESE REAGENTS?

• GO TO www.vetimm.org FOR EMAIL ADDRESS OF SPECIES COORDINATORS

• KINGFISHER BIOTECH @ www.kingfisherbiotech.com

• From Cornell University Animal HealthDiagnostic Cntr http://ahdc.vet.cornell.edu/sects/Serol/

http://ahdc.vet.cornell.edu/sects/Serol/

The Animal Health Diagnostic Center is the NY State Veterinary Diagnostic Laboratory

Equine 5-plex cytokine assay