Mimotopes Pty Ltd
Mimotopes Pty Ltd
News Article | May 10, 2017
Patients who were included in the study all had Goodpasture disease and fulfilled the following key diagnostic criteria: (1) serum anti-α3(IV)NC1 IgG by enzyme-linked immunosorbent assay (ELISA), (2) linear IgG staining of the GBM and (3) necrotizing and crescentic glomerulonephritis. HLA-DR15 typing of patients was done by monoclonal antibody staining (BIH0596, One Lambda) and flow cytometry. Blood from HLA-typed healthy humans was collected via the Australian Bone Marrow Donor Registry. HLA-DR15, HLA-DR1 and HLA-DR15/DR1 donors were molecularly typed and were excluded if they expressed DQB1*03:02, which is potentially weakly associated with susceptibility to anti-GBM disease2. Studies were approved by the Australian Bone Marrow Donor Registry and Monash Health Research Ethics Committees, and informed consent was obtained from each individual. Mouse MHCII deficient, DR15 transgenic mice and mouse MHCII deficient, DR1 transgenic mice were derived from existing HLA transgenic colonies and intercrossed so that they were on the same background as previously described4. The background was as follows: 50% C57BL/10, 43.8% C57BL/6, 6.2% DBA/2; or with an Fcgr2b−/− background: 72% C57BL/6, 25% C57BL/10 and 3% DBA/2. To generate mice transgenic for both HLA-DR15 and HLA-DR1, mice transgenic for either HLA-DR15 or HLA-DR1 were intercrossed. FcγRIIb intact HLA transgenic mice and cells were used for all experiments, except those in experimental Goodpasture disease, where Fcgr2b−/− HLA transgenic strains were used. While DR15+ mice readily break tolerance to α3(IV)NC1 when immunized with human α3 or mouse α3 , renal disease is mild4. As genetic changes in fragment crystallizable (Fc) receptors have been implicated in the development of nephritis in rodents and in humans18, Fcgr2b−/− HLA transgenic strains were used when end organ injury was an important endpoint. For in vitro experiments, cells from either male or female mice were used. For in vivo experiments both male and female mice were used, for immunization aged 8–12 weeks and for the induction of experimental Goodpasture disease aged 8–10 weeks. Experiments were approved by the Monash University Animal Ethics Committee (MMCB2011/05 and MMCB2013/21). HLA-DR15-α3 and HLA-DR1-α3 were produced in High Five insect cells (Trichoplusia ni BTI-Tn-5B1-4 cells, Invitrogen) using the baculovirus expression system essentially as described previously for HLA-DQ2/DQ8 proteins19, 20. Briefly, synthetic DNA (Integrated DNA Technologies, Iowa, USA) encoding the α- and β-chain extracellular domains of HLA-DR15 (HLA-DR1A*0101, HLA-DRB1*15:01), HLA-DR1 (HLA-DR1A*0101, HLA-DRB1*01:01) and the α3 peptide were cloned into the pZIP3 baculovirus vector19, 20. To promote correct pairing, the carboxy (C) termini of the HLA-DR15 and HLA-DR1 α- and β-chain encoded enterokinase cleavable Fos and Jun leucine zippers, respectively. The β-chains also encoded a C-terminal BirA ligase recognition sequence for biotinylation and a poly-histidine tag for purification. HLA-DR15-α3 and HLA-DR1-α3 were purified from baculovirus-infected High Five insect cell supernatants through successive steps of immobilized metal ion affinity (Ni Sepharose 6 Fast-Flow, GE Healthcare), size exclusion (S200 Superdex 16/600, GE Healthcare) and anion exchange (HiTrap Q HP, GE Healthcare) chromatography. For crystallization, the leucine zipper and associated tags were removed by enterokinase digestion (Genscript, New Jersey, USA) further purified by anion exchange chromatography, buffer exchanged into 10 mM Tris, pH 8.0, 150 mM NaCl and concentrated to 7 mg ml−1. Purified HLA-DR15-α3 and HLA-DR1-α3 proteins were buffer exchanged into 10 mM Tris pH 8.0, biotinylated using BirA ligase and tetramers assembled by addition of Streptavidin-PE (BD Biosciences) as previously described19. In mice, 107 splenocytes or cells from kidneys were digested with 5 mg ml−1 collagenase D (Roche Diagnostics, Indianapolis, Indiana, USA) and 100 mg ml−1 DNase I (Roche Diagnostics) in HBBS (Sigma-Aldrich) for 30 min at 37 °C, then filtered, erythrocytes lysed and the CD45+ leukocyte population isolated by MACS using mouse CD45 microbeads (Miltenyi Biotec); they were then surface stained with Pacific Blue-labelled anti-mouse CD4 (BD), antigen-presenting cell (APC)-Cy7-labelled anti-mouse CD8 (BioLegend) and 10 nM PE-labelled tetramer. Cells were then incubated with a Live/Dead fixable Near IR Dead Cell Stain (Thermo Scientific), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-mouse Foxp3 antibody (FJK16 s). To determine Vα2 and Vβ6 usage, cells were stained with PerCP/Cy5.5 anti-mouse Vα2 (B20.1, Biolegend) and antigen-presenting cell labelled anti-mouse Vβ6 (RR4-7, Biolegend). For each mouse a minimum of 100 cells were analysed. The tetramer+ gate was set on the basis of the CD8+ population. In humans, 3 × 107 white blood cells were surface stained with BV510-labelled anti-human CD3 (BioLegend), Pacific Blue-labelled anti-human CD4 (BioLegend), PE-Cy7-labelled anti-human CD127 (BioLegend), FITC-labelled anti-human CD25 (BioLegend) and 10 nM PE-labelled tetramer. Then, cells were incubated with a Live/Dead fixable Near IR Dead Cell Stain (Life Technologies), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-human Foxp3 antibody (150D). The tetramer+ gate was set on the basis of the CD3+CD4− population. As validation controls, we found that HLA-DR1-α3 tetramer+ cells did not bind to HLA-DR1-CLIP tetramers (data not shown). The human α3 peptide (GWISLWKGFSF), the mouse α3 peptide (DWVSLWKGFSF) and control OVA peptide (ISQAVHAAHAEINEAGR) were synthesized at >95% purity, confirmed by high-performance liquid chromatography (Mimotopes). Recombinant murine α3(IV)NC1 was generated using a baculovirus system21 and recombinant human α3(IV)NC1 expressed in HEK 293 cells22. The murine α3(IV)NC1 peptide library, which consists of 28 20-amino-acid long peptides overlapping by 12 amino acids, was synthesized as a PepSet (Mimotopes). To measure peptide specific recall responses, IFN-γ and IL-17A ELISPOTs and [3H]thymidine proliferation assays were used (Mabtech for human ELISPOTs and BD Biosciences for mouse ELISPOTs). To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in patients with Goodpasture disease, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from peripheral blood mononuclear cells of patients with Goodpasture disease (frozen at the time of presentation) by magnetic bead separation (Miltenyi Biotec) then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 2 × 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, α3 (10 μg ml−1) or whole recombinant human α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% male AB serum, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) (Sigma-Aldrich). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in DR15+ transgenic mice, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from pooled spleen and lymph node cells of DR15+ transgenic mice, immunized with mouse α3 10 days previously by magnetic bead separation. They were then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, mouse α3 (10 μg ml−1), human α3 (10 μg ml−1), whole recombinant mα3(IV)NC1 (10 μg ml−1) or whole recombinant hα3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To determine the immunogenic portions of α3(IV)NC1, mice were immunized subcutaneously with peptide pools (containing α3 amino acids 1–92, 81–164, or 153–233; 10 μg per peptide per mouse), the individual peptide or in some experiments mα3 at 10 μg per mouse in Freund’s complete adjuvant (Sigma-Aldrich). Draining lymph node cells were harvested 10 days after immunization and stimulated in vitro (5 × 105 cells per well) with no antigen, peptide (10 μg ml−1) or whole α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml streptomycin). For [3H]thymidine proliferation assays, cells were cultured in triplicate for 72 h with [3H]thymidine added to culture for the last 16 h. To measure human α3 - or mouse α3 -specific responses in CD4+ T cells from naive transgenic mice or blood of healthy humans, we used a modification of a previously published protocol23. One million CD4+ T cells were cultured with 106 mitomycin-treated CD4-depleted splenocytes for 8 days in 96-well plates with or without 100 μg ml−1 of human α3 or mouse α3 . T cells were depleted from mouse cultures by sorting out CD4+CD25+ and in humans by sorting out CD4+CD25hiCD127lo cells using antibodies and a cell sorter. Cytokine secretion was detected in the cultured supernatants by cytometric bead array (BD Biosciences) or ELISA (R&D Systems). To determine proliferation, magnetically separated CD4+ T cells were labelled with CellTrace Violet (CTV; Thermo Scientific) before culture. To measure the expansion of T cells, mice were immunized with 100 μg of α3 emulsified in Freund’s complete adjuvant, then boosted 7 days later in Freund’s incomplete adjuvant. Draining lymph node cells were stained with the HLA-DR15-α3 tetramer, CD3, CD4, CXCR5, PD-1, CD8 and Live/Dead Viability dye. To determine the potency of HLA-DR1-α3 tetramer+ T cells, 106 cells per well of CD4+CD25− T effectors isolated by CD4+ magnetic beads and CD25− cell sorting from naive DR15+DR1+ mice were co-cultured with CD4+CD25+ T cells with or without depletion of HLA-DR1-α3 tetramer+ T cells from DR1+ mice at different concentrations: 0, 12.5 × 103, 25 × 103, 50 × 103 and 100 × 103 cells per well in the presence of 106 CD4-depleted mitomycin C-treated spleen and lymph node cells from DR15+DR1+mice in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) containing 100 μg ml−1 of mouse α3 . To determine proliferation, the CD4+CD25− T effector cells were labelled with CTV before culture. Cells were cultured in triplicate for 8 days in 96-well plates. HLA transgenic mice, on an Fcgr2b−/− background, were immunized with 100 μg of α3 or mα3 subcutaneously on days 0, 7 and 14, first in Freund’s complete, and then in Freund’s incomplete, adjuvant. Mice were killed on day 42. Albuminuria was assessed in urine collected during the last 24 h by ELISA (Bethyl Laboratories) and expressed as milligrams per micromole of urine creatinine. Blood urea nitrogen and urine creatinine were measured using an autoanalyser at Monash Health. Glomerular necrosis and crescent formation were assessed on periodic acid-Schiff (PAS)-stained sections; fibrin deposition using anti-murine fibrinogen antibody (R-4025) and DAB (Sigma); CD4+ T cells, macrophages and neutrophils were detected using anti-CD4 (GK1.5), anti-CD68 (FA/11) and anti-Gr-1 (RB6-8C5) antibodies. The investigators were not blinded to allocation during experiments and outcome assessment, except in histological and immunohistochemical assessment of kidney sections. To deplete regulatory T cells, mice were injected intraperitoneally with 1 mg of an anti-CD25 monoclonal antibody (clone PC61) or rat IgG (control) 2 days before induction of disease. In these experiments, mice were randomly assigned to receive control or anti-CD25 antibodies. Individual DR15-α3 -specific CD4+ T cells were sorted into wells of a 96-well plate. Multiplex single-cell reverse transcription and PCR amplification of TCR CDR3α and CDR3β regions were performed using a panel of TRBV- and TRAV-specific oligonucleotides, as described24, 25. Briefly, mRNA was reverse transcribed in 2.5 μl using a Superscript III VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (containing 1× Vilo reaction mix, 1× superscript RT, 0.1% Triton X-100), and incubated at 25 °C for 10 min, 42 °C for 120 min and 85 °C for 5 min. The entire volume was then used in a 25 μl first-round PCR reaction with 1.5 U Taq DNA polymerase, 1× PCR buffer, 1.5 mM MgCl , 0.25 mM dNTPs and a mix of 25 mouse TRAV or 40 human TRAV external sense primers and a TRAC external antisense primer, along with 19 mouse TRBV or 28 human TRBV external sense primers and a TRBC external antisense primer (each at 5 pmol μl−1), using standard PCR conditions. For the second-round nested PCR, a 2.5 μl aliquot of the first-round PCR product was used in separate TRBV- and TRAV-specific PCRs, using the same reaction mix described above; however, a set of 25 mouse TRAV or 40 human TRAV internal sense primers and a TRAC internal antisense primer, or a set of 19 mouse TRBV or 28 human TRBV internal sense primers and a TRBV internal antisense primer, were used. Second-round PCR products were visualized on a gel and positive reactions were purified with ExoSAP-IT reagent. Purified products were used as template in sequencing reactions with internal TRAC or TRBC antisense primers, as described. TCR gene segments were assigned using the IMGT (International ImMunoGeneTics) database26. In mouse experiments, three mice were pooled per HLA and the number of sequences obtained were as follows. For TRAV: DR15, n = 81; DR1 n = 84; for TRBV: DR15, n = 100; DR1 n = 87; for TRAJ: DR15, n = 81; DR1 n = 84; and for TCR beta joining (TRBJ): DR15, n = 100; DR1 n = 87. Red-blood-cell-lysed splenocytes from DR1+ and DRB15+DR1+ mice were sorted on the basis of surface expression of CD4 and CD25 and being either DR1-α3 tetramer positive or negative into three groups: (1) CD4+CD25−HLA-DR1-α3 tetramer− T cells; (2) CD4+CD25+HLA-DR1-α3 tetramer− T cells; and (3) CD4+CD25+HLA-DR1-α3 tetramer+ T cells. A minimum of 1,000 cells were sorted. Immediately after sorting, the RNA was isolated and complementary DNA (cDNA) generated using a Cells to Ct Kit (Ambion) followed by a preamplification reaction using Taqman Pre Amp Master Mix (Applied Biosystems), which preamplified the following cDNAs: Il2ra, Foxp3, Ctla4, Tnfrsf18, Il7r, Sell, Pdcd1, Entpd1, Cd44, Tgfb3, Itgae, Ccr6, Lag3, Lgals1, Ikzf2, Tnfrsf25, Nrp1, Il10. The preamplified cDNA was used for RT–PCR reactions in duplicate using Taqman probes for the aforementioned genes. Each gene was expressed relative to 18S, logarithmically transformed and presented as a heat map. The Epstein-Barr-virus-transformed human B lymphoblastoid cell lines IHW09013 (SCHU, DR15-DR51-DQ6) and IHW09004 (JESTHOM, DR1-DQ5) were maintained in RPMI (Invitrogen) supplemented with 10% FCS, 50 IU ml−1 penicillin and 50 μg ml−1 streptomycin. Confirmatory tissue typing of these cells was performed by the Victorian Transplantation and Immunogenetics Service. The B-cell hybridoma LB3.1 (anti-DR) was grown in RPMI-1640 with 5% FCS at 37 °C and secreted antibody purified using protein A sepharose (BioRad). HLA-DR-presented peptides were isolated from naive DR15+Fcgr2b+/+ or DR1+Fcgr2b+/+ mice. Spleens and lymph nodes (pooled from five mice in each group) or frozen pellets of human B lymphoblastoid cell lines (triplicate samples of 109 cells) were cryogenically milled and solubilized as previously described12, 27, cleared by ultracentrifugation and MHC peptide complexes purified using LB3.1 coupled to protein A (GE Healthcare). Bound HLA complexes were eluted from each column by acidification with 10% acetic acid. The eluted mixture of peptides and HLA heavy chains was fractionated by reversed-phase high-performance liquid chromatography as previously described10. Peptide-containing fractions were analysed by nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) using a ThermoFisher Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) operated as described previously10. LC–MS/MS data were searched against mouse or human proteomes (Uniprot/Swissprot v2016_11) using ProteinPilot software (SCIEX) and resulting peptide identities subjected to strict bioinformatic criteria including the use of a decoy database to calculate the false discovery rate28. A 5% false discovery rate cut-off was applied, and the filtered data set was further analysed manually to exclude redundant peptides and known contaminants as previously described29. The mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE30 partner repository with the data set identifier PXD005935. Minimal core sequences found within nested sets of peptides with either N- or C-terminal extensions were extracted and aligned using MEME (http://meme.nbcr.net/meme/), where motif width was set to 9–15 and motif distribution to ‘one per sequence’31. Graphical representation of the motif was generated using IceLogo32. Crystal trials were set up at 20 °C using the hanging drop vapour diffusion method. Crystals of HLA-DR15-α3 were grown in 25% PEG 3350, 0.2 M KNO and 0.1 M Bis-Tris-propane (pH 7.5), and crystals of HLA-DR1-α3 were grown in 23% PEG 3350, 0.1 M KNO , and 0.1 M Bis-Tris-propane (pH 7.0). Crystals were washed with mother liquor supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen before data collection. Data were collected using the MX1 (ref. 33) and MX2 beamlines at the Australian Synchrotron, and processed with iMosflm and Scala from the CCP4 program suite34. The structures were solved by molecular replacement in PHASER35 and refined by iterative rounds of model building using COOT36 and restrained refinement using Phenix37 (see Extended Data Table 2 for data collection and refinement statistics). No statistical methods were used to predetermine sample size. For normally distributed data, an unpaired two-tailed t-test (when comparing two groups). For non-normally distributed data, non-parametric tests (Mann–Whitney U-test for two groups or a Kruskal–Wallis test with Dunn’s multiple comparison) were used. Statistical analyses, except for TCR usage, was by GraphPad Prism (GraphPad Software). For each TCR type/region (TRAV, TRBV, TRAJ, TRBJ), we compared the TCR distribution (frequencies of different TCRs) between DR15 and DR1 using Fisher’s exact test. This was applied both to mice and to human samples. The P values associated with those TCR distributions are indicated above the pie-charts. To correct for multiple testing for individual TCRs, we used Holm’s method. *P < 0.05, **P < 0.01, ***P < 0.001. The data that support the findings of this study are available from the corresponding authors upon request. Self-peptide repertoires have been deposited in the Proteomics Identifications Database archive with the accession code PXD005935. Structural information has been deposited in the Protein Data Bank under accession numbers 5V4M and 5V4N.
Rhodes D.I.,Avexa Ltd |
Rhodes D.I.,JDJ Bioservices Ltd |
Peat T.S.,CSIRO |
Vandegraaff N.,Avexa Ltd |
And 8 more authors.
ChemBioChem | Year: 2011
An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Ede N.J.,Mimotopes Pty Ltd. |
Hill J.,Agency for Science, Technology and Research Singapore |
Joy J.K.,Agency for Science, Technology and Research Singapore |
Ede A.-M.,Mimotopes Pty Ltd. |
Koppens M.L.,Mimotopes Pty Ltd.
Journal of Peptide Science | Year: 2012
Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single-stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid-phase synthesis of a library of 400 C-terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies. © 2012 European Peptide Society and John Wiley & Sons, Ltd.