Immunological Toolbox

Key targets

• IL-15 (pAb, mAbs)
• IL-6  (pAb, mAbs)
• TNF (pAb, mAbs)
• RANTES (pAb, mAbs)

Summary of progress

Other targets

• Screen existing mAbs for cross-reactivity with horses and characterise

Summary of progress

 

Progress on key targets for horses
Updated 22 July 2009

IL-15
Prokaryotic: A 510bp (EcoRI & BamHI) fragment was amplified from the pcDNA3IGHG1-IL15 plasmid and subcloned into pGEX3X. The construct was transformed into E.coli codon plus cells and fusion protein expressed. The insoluble fusion protein was used to immunise two sheep and antisera are frozen. The antisera were positive by Western blot using a lysate from bacteria transformed with IL15-pGEX3X plasmid and antiserum specific for GST as a positive control but negative by flow cytometry using Cos 7 cells transiently transfected with pcDNA3IGHG1-IL-15 plasmid.
Eukaryotic: A 511bp (EcoRI & BamHI) fragment was amplified from lymph node cells and cloned into pcDNA3IGHG1. Four coding changes (frame shifts) were identified. Therefore, the correct fragment was re-cloned. Transfection was not carried out before the end of the project, but the products to date have been made available for other workers.

IL-6
Prokaryotic: A 572bp (BamHI & EcoRI) and 571bp (NdeI & XhoI) fragment was cloned into pGEX3X and pET21A respectively. Protein expression was achieved successfully with pET21A transformed into E. coli (BL21-DE3) but the product was insoluble. Therefore the fusion protein was used to immunise two sheep. Polyclonal antisera were generated and characterised as follows: bacterial lysates transformed with pET21-IL6 were recognised by the antisera at the predicted size in Western blotting. Labelling of transiently transfected Cos 7 cells with the pcDNA3IGHG1-IL6 plasmid using the antiserum produced 13% +ve cells by indirect immunofluorescence and flow cytometry.
Eukaryotic: A 652bp fragment was subcloned (BamHI & NheI) into pcDNA3IGHG1 and transfected into CHO-K1 cells by Dr Wagner. Cells are frozen in liquid nitrogen.

Tumour necrosis factor alpha (TNFα)
Prokaryotic: A 721bp (BamHI & EcoRI) fragment was cloned into pGEX3X and transformed into E. coli Codon Plus cells. Soluble protein was expressed which was partially degraded but used to immunise two sheep. The pooled antiserum detected recombinant protein of the predicted size in Western blot. Competition and inhibition ELISAs were developed. The antiserum blocked a bioassay which measured TNFα activity. A capture ELISA detected TNFα in nasal swab extracts taken from horses during the first week after infection with equine influenza virus. No staining of formalin fixed lymph node cells collected from horses after infection with the bacterium S. equi or from arthritic joint tissue could be detected by indirect immunohistochemistry using this antiserum. Similarly, flow cytometry analysis of the antiserum using mitogen stimulated lymphocytes was negative. These results are consistent with the antibody reagent detecting TNF-α induced in vivo following virus infection.
Eukaryotic: A 720bp fragment (BamHI & NheI) was cloned into pcDNA3IGHG1 and transfected into CHO-K1 cells. The cells were expanded until 99% were positive by flow cytometry detecting IgHG1 and frozen.

Chemokine ligand 5 (CCL5; RANTES)
Prokaryotic: A 225bp (NheI & XhoI) or 227bp (BamHI & EcoRi) fragment of RANTES was amplified from mitogen activated PBMC, cloned into pET21A and pGEX3X respectively and transformed into codon plus E. coli for expression. Expression failed on several occasions and so efforts were re-directed to focus on eukaryotic and baculovirus expression.
Eukaryotic: a 299bp fragment was amplified from mitogen activated PBMC and cloned into pcDNA3IGHG1. The plasmid was stably transfected into CHO-K1 cells and stocks are frozen in liquid nitrogen.
Insect: a 299bp fragment was amplified from the pcDNA3IGHG1-RANTES plasmid and cloned into pFASTBacHTa (Invitrogen) with EcoRI and HindIII restriction enzymes sites. A baculoviral stock was produced in sf9 cells and frozen at -70°C for future purification.

 

Progress on other targets for horses
Updated 22 July 2009

Screening of existing mAbs for crossreactivity with horses
In collaboration with Dr Falko Steinbach of the Institute for Zoo and Wildlife Research, Berlin, we screened 379 anti-human leukocyte monoclonal antibodies for cross reactivity with equine leukocytes. Approximately 4% of anti-human mAb cross-reacted with equine leukocytes and of these, mAb directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172 were the most effective (cross-reactivity was anticipated for these molecules based on similarities between the human and equine staining pattern). See Steinbach et al 2007 and Ibrahim et all 2007 in publications list.
A mAb (CC302), known to recognize bovine IFNg, was characterized and shown to identify equine IFNg. Flow cytometric and ELIspot assays to quantify the number of lymphocytes which express this intracellular cytokine were developed. These assays were applied to characterise the equine cellular immune response to equine herpesvirus-1 and equine influenza virus after experimental and field infections and vaccination with new generation products. See Paillot et al 2005, 2006 in the publications list.

PCR test to identify horses with the MHC class I B2 allele
Primers specific for the B2 allele were designed and used in a PCR to amplify a product from cDNA derived from B2 homozygous animals. This was in collaboration with Prof Doug Antczak, Cornell University and Dr Shirley Ellis, IAH, Compton. The PCR test was applied to 40 Thoroughbred horses. A total of 12 putatively homozygous animals were identified and the PCR products have been cloned and at least 15 clones of each sequenced to determine genetic variability. The test shows promise as a molecular method to identify the B2 allele which is expressed on the A3 serological haplotype. It is currently being evaluated in the selection of ponies for vaccine trials to study the cellular immune response to a CTL target protein of EHV-1. If successful, this test will assist in refining the use of experimental animals.

Construction of tetramers involving equine MHC class I B2 and EHV-1 CTL target peptide encoded by gene 64
This work was carried out in collaboration with Prof Paul Lunn & Dr Gisela Hussey, Colorado State University, USA. To identify the CTL target peptide(s) encoded by EHV-1 gene 64, the sequence (~4,500bp) has been fragment cloned into pcDNA6/V5 and the target sequence has been verified. Twelve fragments were transiently transfected into Cos 7 cells and expression confirmed by detection of V5 in indirect immunofluorescence microscopy and / or flow cytometry. This work is ongoing through grant funding to Professor Lunn's group.

CD14
Prokaryotic: A 1072bp (NdeI & XhoI) and 1071bp (BamHI & EcoRI) fragment of CD14 was amplified from a plasmid kindly donated by Dr C Bryant (University of Cambridge) and cloned into pET21A and pGEX3X respectively and transformed into E.coli (Codon+ or DL21-DE3). Soluble protein was induced from the pGEX3X construct, purified with glutathione S transferase beads and the fusion protein (GST would not cleave) used to immunise two sheep. Polyclonal antisera were positive by Western blot of Cos 7 cells transiently transfected with pcDNA3IGHG1-CD14, chicken CD14 (a kind gift from Dr Pete Kaiser, IAH, Compton) and ELISA. Blood monocytes were negative for surface expression of CD14 by flow cytometry and Western blotting.
Eukaryotic: A 1132bp (NheI & BamHI) fragment of CD14 was cloned into pcDNA3/IGHG1, expressed and transfected into CHO-K1 cells using lipofectamine. A cell line (23% of cells positive by intracellular staining) has been cryopreserved.

Granzyme B
Prokaryotic: A 709bp (BamHI & EcoRI) fragment was amplified from PBMC and cloned into pGEX3X and transformed into E. coli Codon plus cells. Induction of protein was unsuccessful.
Eukaryotic: A 713bp (NheI & BamHI) fragment was amplified from PBMC and cloned into pcDNA3IGHG1. Four coding changes were noted but the plasmid was transfected into CHO-K1 cells using lipofectamine. A total of 70% positive cells were detected by intracellular flow cytometry using a monoclonal antibody specific for equine IgG1. Stocks of Granzyme B transfectants have been cryopreserved in liquid nitrogen.

IL-17
Prokaryotic: A 412 bp (Nde1 & Xho1) product was amplified from activated lymphocytes and cloned into pET21a. No further experiments were conducted within the timeframe of the project.
Eukaryotic: A product could not be amplified using cDNA from a variety of template sources.
Insect: A product was amplified from pET21A-IL-17 and subcloned into pFastBacHTa. Recombinant baculovirus was expressed in sf9 cells and the supernatant has been frozen at -70°C for future purification.

Lactoferrin
Prokaryotic: A 2085bp (NheI & HindIII) and 2083bp (EcoRV) fragment was cloned into pET21A and pGEX3X respectively. The former was transformed into E. coli BL21A-DE3 and although low levels of protein were expressed, it could not be purified.
Eukaryotic: Due to the difficulty of cloning into prokaryotic expression vectors, cloning was not attempted in this system, but all efforts directed to insect expression.
Insect: A 497bp fragment was subcloned into pFastBacHTa and successfully expressed in sf9 cells. Recombinant baculovirus stock has been frozen at -70°C.

Transferrin
Prokaryotic: Two fragments, 2082bp (BamHI) and 2090bp (NdeI & NotI) fragment were subcloned into pET21A and pGEX3X respectively. The former was transformed into E. coli BL21-DE3 and insoluble protein was expressed. Although attempts were made to solubilise with urea/guanidine HCl, the protein product could not be purified.
Eukaryotic: A 2141bp fragment (NheI & BamHI) was cloned into pcDNA3IGHG1. Five coding changes were noted. The construct was transfected into CHO-K1 on several occasions, but transfection efficiency was poor. Attempted recloning was unsuccessful.

Interferon β
A 550bp (EcoRI & BamHI) fragment was amplified from equine influenza virus-infected tracheal cells collected one day after experimental infection. The fragment was cloned into pGEX3X and transformed into codon plus E. coli cells. Protein was expressed at low levels but the majority was insoluble, which did not allow purification.

Toll like receptor 4
A 2540bp product was amplified from normal equine placenta and cloned into pcDNA3IGHG1. The construct was transfected into CHO-K1 cells but transfection efficiency was poor as detected by indirect immunofluorescence in flow cytometry. Similarly, supernatant medium was negative for expression of equine IGHG1 by ELISA.

Toll like receptor 2
A 500bp fragment (BamHI & EcoRI) was amplified from leukocytes and cloned into pGEX3X. A product was expressed in codon plus cells, but at a very low level. Several unsuccessful attempts to increase expression were made.

iNOS
A 454bp fragment (BamH1 & EcoR1) was amplified from leukocytes and cloned into pGEX3X. When the plasmid was transformed into codon plus cells, only the GST tag was expressed, despite the plasmid having correct sequence

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