MATERIALS AND METHODS FOR INDUCING REGULATORY T CELLS
20210355176 · 2021-11-18
Inventors
- Richard Maizels (Glasgow, Lanarkshire, GB)
- Danielle Smyth (Glasgow, Lanarkshire, GB)
- Henry McSorley (Edinburgh, Lothian, GB)
Cpc classification
A61K35/17
HUMAN NECESSITIES
C12N5/0637
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
Abstract
The present invention concerns a structurally distinct immunosuppressive mimic of TGF-β that is a potent inducer of murine and human regulatory T cells and provides a therapeutic agent for the treatment of inflammatory disorders. Disclosed herein is a novel parasite TGF-β mimic which fully replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing Foxp3.sup.+ Treg in both murine and human CD4.sup.+ T cells. This TGF-β mimic shares no homology to mammalian TGF-β or other members of the TGF-β family, but s distinctly related to the component control protein (CCP) superfamily.
Claims
1. A polypeptide comprising amino acid sequence of domain 1 and domain 3 of a TGM protein and having TGF-β receptor agonist activity.
2. A polypeptide according to claim 1 further comprising a linker amino acid sequence of between 10 and 150 amino acids positioned between the amino acid sequence of domain 1 and the amino acid sequence of domain 3.
3. A polypeptide sequence according to claim 2 wherein the linker amino acid sequence is domain 2 of a TGM protein.
4. A polypeptide comprising amino acid sequence 19 to 228 of a TGM protein and having TGF-β receptor agonist activity.
5. A polypeptide according to claim 1 comprising domains 1 to 5 of a TGM protein wherein domains 1, 2 and 3 have at least 90% sequence identity to TGM, TGM-a or TGM-b; and wherein domains 4 and 5 have at least 60% sequence identity to TGM, TGM-a or TGM-b.
6. A polypeptide according to claim 1 wherein the polypeptide comprises one or more conservative amino acid substitutions.
7. A polypeptide according to claim 1 comprising an amino acid sequence selected from TGM, TGM-a, TGM-b, and TGM-d as provided in Table 1.
8. A nucleic acid sequence encoding a polypeptide according to claim 7.
9. An expression vector comprising a nucleic acid sequence according to claim 8.
10. An expression vector according to claim 9 wherein the nucleic acid sequence is operably linked to a promoter.
11. An expression vector according to claim 9 which is a viral vector.
12. A host cell comprising a nucleic acid sequence according to claim 8 or an expression vector containing said nucleic acid sequence.
13. An immunosuppressive agent comprising a TGM polypeptide according to claim 7, or a nucleic acid sequence encoding said polypeptide, said nucleic acid optionally being present in an expression vector.
14. An immunosuppressive agent according to claim 13 wherein said TGM polypeptide forms a complex with one or more further polypeptide sequences as a fusion protein.
15. A pharmaceutical composition comprising an immunosuppressive agent according to claim 13 in a physiologically acceptable carrier.
16. (canceled)
17. (canceled)
18. (canceled)
19. A method of treating an inflammatory disorder or disease in a subject comprising administering a therapeutic amount of a pharmaceutical composition according to claim 15 to said subject in need thereof.
20. A method according to claim 19 wherein said inflammatory disorders or diseases are selected from the group consisting of allergic diseases, hay fever, food allergies, atopic dermatitis, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, celiac disease, immune-mediated inflammatory diseases, ankylosing spondylitis, vasculitis, arthritis, hepatitis, chronic inflammatory demyelinating polyneuropathy, rheumatoid arthritis, colitis, Crohn's disease, ulcerative colitis, erythema, rheumatism, inflammatory bowel disease, pelvic inflammatory disease, thyroiditis, myopathy, myositis, Behcet's disease, psoriasis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, allografts reactivity leading to rejection of skin and/or organ transplants, and graft-versus-host disease following bone marrow transplantation.
21. A method of converting peripheral T cells into regulatory T cells (Treg cells) for use in treating inflammatory diseases, said method comprising contacting a sample of peripheral T cells with a polypeptide of claim 1, culturing said cells with said polypeptide in culture medium, and collecting converted Treg cells from said culture medium.
22. A method according to claim 21 wherein the peripheral T cells are obtained from an individual suffering from an inflammatory disorder or disease.
23. A method according to claim 21 further comprising the step of preparing a pharmaceutical composition comprising said converted Treg cells.
24. A method according to claim 22 further comprising administering said converted Treg cells to said individual suffering from an inflammatory disorder or disease.
25. A method according to claim 24 wherein the inflammatory disorders or diseases are selected from the group consisting of allergic diseases, hay fever, food allergies, atopic dermatitis, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, celiac disease, immune-mediated inflammatory diseases, ankylosing spondylitis, vasculitis, arthritis, hepatitis, chronic inflammatory demyelinating polyneuropathy, rheumatoid arthritis, colitis, Crohn's disease, ulcerative colitis, erythema, rheumatism, inflammatory bowel disease, pelvic inflammatory disease, thyroiditis, myopathy, myositis, Behcet's disease, psoriasis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, allografts reactivity leading to rejection of skin and/or organ transplants, and graft-versus-host disease following bone marrow transplantation.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0113]
[0117]
[0123]
[0129]
[0130] Mass spectrometric profiles of candidate proteins. Protein abundance profiles for all candidate proteins found in both gel-filtration (upper panels) and ion exchange (lower panels) fractions with peak TGF-β activity, except for Hp_103161_IG00349_L1408 shown in
[0131]
[0132] Expression of recombinant TGM.
[0133] A. Secreted purified recombinant TGM from transfected HEK293T cells analysed by SDS-PAGE electrophoresis and Coomassie Blue staining.
[0134] B. Western blot analysis with anti-penta-His antibody
[0135]
[0136] TGM induces de novo Foxp3 expression in murine and human CD4+ T cells and induces greater Foxp3 expression than hTGF-β at high concentrations. [0137] A, B. CD4+CD25-GFP-CD62L.sup.hi murine naïve T cells were stimulated with plate-bound anti-CD3/CD28 for 4 days in culture with 100 U/ml IL-2 and variable concentrations of TGM or hTGF-β1, before flow cytometric analysis of CD4, CD25 and Foxp3 expression; 2 technical replicates per concentration; representative of 4 independent experiments. A: percentage of CD25.sup.+ Foxp3.sup.+ cells among total CD4.sup.+ cells; B, Mean fluorescence intensity (MFI) of Foxp3 among Foxp3.sup.+ cells. [0138] C-E. Foxp3 induction in the same conditions as A, in the presence of SB431542 inhibitor (C) or pan-vertebrate anti-TGF-β (D, E); 2 technical replicates per concentration; representative of 3 independent experiments. [0139] F-H. Human peripheral blood mononuclear cells were separated from red blood cells over a Ficoll gradient and CD4.sup.+ T cells isolated by MACS positive selection. Isolated cells were cultured at 37° C. for 96 hours with a 1:1 ratio of CD3/CD28 Dynabeads® and variable concentrations of hTGF-β1 or TGM. Induction of Tregs from human peripheral blood monuclear cells (or just PBMCs if the terms has been used before) F: Representative flow cytometry plots (CD4.sup.+ population shown) MACS CD4+ positive selected PBMCs stimulated with Hp-TGM, hTGF-β or II-2 respectively; G: percentage of CD25.sup.+ Foxp3.sup.+ cells among total CD4.sup.+ cells; H, MFI of Foxp3 among Foxp3.sup.+ cells; 2 technical replicates per concentration; representative of 2 independent experiments.
[0140]
[0141] TGM drives immune regulation in vitro and in vivo. [0142] A. Murine Foxp3.sup.+ Treg are functionally suppressive in vitro. Foxp3.sup.+ Treg were generated in vitro (as in
[0151]
[0152] Protective effects of TGM in the T cell transfer mouse model of colitis. A.
[0153] Experimental outline and summary of time points for insertion of osmotic minipumps (d-1), transfer of T cells (d0) and analysis of intestinal inflammation (d60). B. Summary of inflammatory response in intestinal tissue, assessed on 15-point histopathological scale measuring crypt architecture, ulceration, crypt abscesses, goblet cell loss, mucosal inflammatory infiltration and submucosal inflammatory infiltration. C. Example histopathological staining with haematoxylin and eosin (H&E) in sections of intestinal tissue from mice receiving ovalbumin (OVA) or TGM.
[0154]
[0155] Domain structure of TGM variants -TGM, TGM-a and TGM-d have been verified as active.
[0156]
[0157] Alignment of TGM Sequences which are active (TGM, TGM-a and TGM-d) or inactive (TGM-b).
[0158]
[0159] TGM truncation constructs, made to express protein containing 1-4 of the 5 domains as indicated. Truncated nucleotide sequences corresponding to the indicated domains were constructed in pSecTag plasmids and used to transfect HEK cells; supernatant from transfected cells was analysed by SDS-PAGE and Western Blot (Lower Right Hand panel), showing that all truncated proteins were expressed although levels of expression of Domain 1 and Domain 5 were relatively low (position on the Western Blot indicated by asterisks). Numerals on the left of the Western Blot denote mol.Math.wt in kDa.
[0160]
[0161] Analysis of activity in truncation constructs.
DETAILED DESCRIPTION
[0162] The inventors have taken a fractionation approach to identify immunomodulatory proteins contained within HES, in order to develop these as potential drugs for use in human inflammatory disease. They fractionated HES by size (by gel filtration chromatography) and by charge (using anion exchange chromatography) to yield 20-30 individual fractions, each of which contained a small pool of molecules. These fractions were then assessed for TGF-β signalling ability using the MFB-F11 TGF-β bioassay described by Tessuer in 2006.sup.25. (see
[0163] Relative abundances of proteins present in all size and charge fractions were assessed by LC-MS/MS mass spectrometry, referenced to an in-house generated transcriptome and proteome of H. polygyrus and HES.
[0164] Using cross-referencing of immunomodulatory activity to relative abundances by both methods of fractionation, the inventors were able to identify a small number (<10) of candidate proteins. The genes encoding these were codon optimised and cloned for expression in HEK293 mammalian cells. Recombinant proteins were tested for TGF-β signalling, using the MFBF11 assay described above. This technique resulted in identification of TGM (TGF-β mimic) which stimulates a stronger signal in said assay than mammalian TGF-β itself (
[0165] TGM can signal through the mammalian TGF-β receptor at concentrations close to that of recombinant active human TGF-β (
[0166] Despite having very similar action to mammalian TGF-β, TGM has no sequence similarity with TGF-β, and has no known homologues in any other organism. The sequence of TGM predicts a 46.9 kDa protein, with 22 cysteine residues, and 5 predicted N-glycosylation sites. The high proportion of cysteine residues may indicate multiple disulphide bonds, which makes the protein particularly stable. Experimental data supports this: TGM heated to 37° C. for 28 days lost no detectable activity. H. polygyrus contains a family of TGM homologues, which the inventors have tested for TGF-β signalling ability. Comparison of sequence similarity with TGF-β signalling will identify minimal motifs required for binding, and lead to the provision of peptide mimetics for development as stable, non-immunogenic, tissue-penetrative drugs.
[0167] Materials and Methods
[0168] Animals. Inbred female C57BL/6J OlaHsd, BALB/c OlaHsd, and Foxp3-GFP reporter mice.sup.26 were used for experiments, aged 6-12 weeks old and bred in-house or purchased from Harlan Laboratories. All animal experiments were performed under UK Home Office licence and supervised by the Veterinary Services at the Universities of Edinburgh and Glasgow.
[0169] Fractionation of HES and Mass Spectrometric Analysis of HES Fractions.
[0170] Heligmosomoides polygyrus Excretory Secretory (HES) products were prepared as described elsewhere.sup.24. HES was separated into 1 ml fractions using an ÄKTApurifier™ (GE Healthcare) using either the Superdex 200 10/300 GL column (GE Healthcare) for gel filtration fractionation or the Mono Q™ 5/50 GL (GE Healthcare) for anion exchange fractionation. The protein concentration of fractions was measured by Pierce BCA Protein assay kit (Thermo Scientific) with 5 μg of each fraction being trypsin digested, analysed using an Orbitrap mass spectrometer and then compared to an in house H. polygyrus transcriptomics database using Mascot. The significance threshold for consideration of proteins was p=0.05. Proteins with a score below 20 were not considered. Scripts written in Python 2.7 were used to analyse the mass spectrometry results shown in
[0171] Recombinant TGM. Recombinant TGM was synthesised as a mammalian codon optimised insert (GeneArt) and cloned into the mammalian expression vector pSECTag2a (Invitrogen). The construct was transfected into HEK293T cells using the calcium chloride transfection method (Promega) and recombinant TGM was purified from culture supernatant by Ni-chelating chromatography (
[0172] Antibodies and Inhibitors. The mouse monoclonal pan-isoform specific TGF-β IgG antibody, 1D11.sup.27, was purchased (BioXCell), as were the inhibitors SB431542 (Tocris Bioscience) and ITD-1 (Tocris Bioscience). The mouse IgG1 myeloma MOPC 31C from ATCC (ECACC-90110707) was used as an isotype control. Smad2/3 antibody (Cell Signaling Technology) and phosphoSmad2 antibody (Cell Signaling Technology) was used as the primary antibody for Western blots, followed by Goat anti rabbit IgG-HRP secondary (BioRad) according to manufacturer's protocols. Recombinant TGM was detected by Western blot using an anti penta-His-HRP conjugate (Qiagen).
[0173] TGF-β Bioassay. The TGF-β bioassay (MFB-F11) developed by Tesseur et al..sup.25 was used with embryonic fibroblasts from TGF-β1.sup.−/− mice stably transfected with a TGF-β-responsive reporter plasmid containing a secreted embryonic alkaline phosphatase reporter gene (SBE-SEAP). MFB-F11 cells were grown in DMEM with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine and supplemented with 15 μg/ml Hygromycin B (Invitrogen), for 3 days. Cells were tested and found to be mycoplasma-free. Confluent cells were detached with trypsin, and resuspended in DMEM with 2.5% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at a concentration of 4×10.sup.5 cells/ml. In 50 μl, 4×10.sup.4 cells were added to each well of a 96-well round-bottomed plate. Serial dilutions of test substances HES (in-house), TGM (in-house), and recombinant human TGF-β1 (R&D Systems) were then added to each well in a volume of 50 μl and incubated for 24 hours at 37° C. Subsequently, 20 μl of supernatant was aspirated from each well, added to an ELISA plate (NUNC) with 180 μl of reconstituted Sigma Fast™ p-nitrophenyl phosphate substrate and incubated at RT in the dark for up to 4 hours. Plates were read on at 405 nm on an Emax precision microplate reader (Molecular Devices).
[0174] Cellular Immunology Assays. Single cell suspensions were made from murine spleen and lymph node specimens by maceration through 70 μm filters (BD) into complete RPMI 1640 (cRPMI) medium containing HEPES (Gibco), supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco), 10% heat-inactivated foetal calf serum (FCS) (Gibco), and 50 nM 2-mercaptoethanol (Gibco). Contaminating red blood cells were removed by resuspending the cells from one spleen in 2 ml of red blood cell lysis buffer (Sigma) and incubating at RT for 2 minutes. Cells were then washed with cRPMI and counted on a haemocytometer by trypan blue exclusion. For human lymphocytes, fresh peripheral blood was obtained by venepuncture of healthy volunteers under a protocol approved by the University of Edinburgh research ethics committee. Blood was collected into heparinised tubes (BD) and immediately diluted 1:1 with PBS, centrifuged over Ficoll-Paque (GE Healthcare) at 400 g for 40 minutes at RT with no brake, and PBMCs recovered from the interface before three further washes in cRPMI at 200 g for 10 minutes (RT). Finally, cells were counted on a haemocytometer in preparation for culture.
[0175] Flow Cytometric Analysis and Cell Sorting. For viability staining, LIVE/DEAD® fixable blue (Life Technologies) was diluted to 1:1000 in PBS; 200 μl was added to each sample of cells, which were then incubated in the dark for 20 min at 4° C. (protected from light) and washed twice in FACS buffer. To prevent non-specific antigen binding, cells were incubated with 50 μl of polyclonal IgG (diluted 1:50 in FACS buffer) for 10 minutes at 4° C. and then washed twice in FACS buffer. All samples were acquired on a BD Biosciences LSR II or LSR Fortessa flow cytometer and analysed using FlowJo software (Tree Star).
[0176] The following FACS antibodies were diluted to an appropriate final concentration in FACS buffer (or permeabilisation buffer (eBioscience) for intracellular antibodies): Anti-CD3-FITC (17A2, Biolegend); anti-CD4-AF700 and -BV650 (RM4-5, Biolegend); anti-CD8-PerCP (53-6.7, Biolegend); anti-CD25-APC (PC61-5, eBioscience); anti-Foxp3-ef450, (FJK-16 s, eBioscience), anti-ROR-gamma(t)-PE (AFKJS-9, eBioscience); anti-Tbet-PerCP-Cyanine (eBio4BIO, eBioscience) to a total volume of 50 μl diluted antibody per 5×10.sup.6 cells. Single stain controls were individually added to one drop of UltraComp eBeads (eBioscience). Samples were incubated for 20 min at 4° C., washed twice in FACS buffer and then resuspended in 200 μl FACS buffer for acquisition of surface marker data directly or further processed for intracellular staining.
[0177] Transcription Factor Staining. For analysis of transcription factors, cells were resuspended in 400 μl fixation/permeabilisation buffer (eBioscience) and incubated at 4° C. for between 1 and 18 hours. Following incubation, cells were resuspended and washed twice in 1 ml permeabilisation buffer (eBioscience). 50 μl of antibody or isotype control (diluted in permeabilisation buffer) was added to each sample. Cells were resuspended by gentle vortex and incubated at room temperature for 30 minutes. Finally, cells were washed in 2 ml of FACS buffer and resuspended in 200 μl FACS buffer for acquisition.
[0178] CD4.sup.+ T Cell Enrichment by Magnetic Sorting. Cells were resuspended in MACS buffer at a volume of 45 μl per 10.sup.7 cells, together with 5 μl of microbeads (L3T4, Miltenyi Biotech), and incubated at 4° C. for 20 minutes. Cells were then washed three times in MACS buffer, centrifuging at 200 g for 5 minutes, and resuspended in MACS buffer at a volume of 50 μl per 10.sup.7 cells. CD4.sup.+ cells were then isolated by performing a positive selection using an AutoMACS (Miltenyi Biotech) automated magnetic column as per the manufacturer's instructions. The positive fraction of cells was then resuspended in MACS buffer and counted.
[0179] Fluorescence-Activated Cell Sorting. CD4.sup.+ cells (freshly isolated or from culture) were enriched by magnetic sorting as above and then incubated with antibodies for surface markers as described above, but with the omission of a viability stain. Following staining, cells were resuspended in MACS buffer at a concentration of 5×10.sup.8 cells per ml. Sorting was performed on a BD FACSAria with a gating strategy of: lymphocytes (by forward and side scatter), single cells and then stained populations, e.g. CD4.sup.+CD25.sup.− Foxp3.sup.−CD62L.sup.hi. Cells were sorted into 2 ml of FCS (Gibco) and a sample from each tube was re-acquired on the FACSAria to assess the purity of each sort.
[0180] Foxp3.sup.+ Treg Induction Assay. A single cell suspension was prepared from the spleen and peripheral lymph nodes of Foxp3-GFP transgenic mice. CD4+CD25-GFP-CD62Lhi cells were then isolated by MACS followed by FACS sorting (see previous sections). Sorted cells were washed twice in complete RPMI and then resuspended in complete RPMI at a concentration of 5×10.sup.5 cells per ml. CD3/CD28-coated 24 well plates (Costar) were prepared by adding 250 μl per well of CD3 and CD28 (eBioscience), both at 2 μg/ml in PBS, incubating for 2 hours at 37° C. and then washing 3 times in PBS. 5×10.sup.5 cells were then added to each well in 1 ml of complete RPMI. Each well was made up to final volume of 2 ml complete RPMI, containing variable concentrations of treatment conditions (eg. TGF-β) and IL-2 (produced in-house) at a final concentration of 100 U/ml. Cells were removed after 96 hours for flow cytometric analysis.
[0181] Treg Suppression Assays. Tregs induced as above were washed in MACS buffer and the CD4.sup.+CD25.sup.+GFP.sup.+ Treg population was isolated by FACS sorting. Responder cells (CD4.sup.+CD25.sup.−GFP.sup.−CD62L.sup.hi) were also isolated from a fresh Foxp3-GFP transgenic mouse. 10.sup.4 responder cells were added to each well of a 96 well round-bottomed plate together with 10.sup.5 irradiated APCs, 2 μg/ml soluble CD3 stimulation and a variable concentration of Treg. Proliferation was assessed after 72 hours by thymidine incorporation.
[0182] Continuous Infusion via Osmotic Minipump. Alzet minipumps (Charles River UK) of 100 μl capacity were selected according to the duration of infusion required for individual experiments (model 1007D—7 days; model—1002—14 days; model 1004—28 days). Minipumps were filled with the substance for infusion (HES, TGM or PBS control) and primed overnight by incubation in PBS at 37° C.
[0183] Under general anaesthesia, abdominal fur was removed by shaving and the skin was prepared with chlorhexidine solution. The peritoneal cavity was accessed through an upper midline incision and the minipump was placed in the right paracolic gutter. Closure was in two layers with 5-0 undyed Vicryl® (Ethicon UK).
[0184] Skin Transplantation. Full-thickness skin transplantation was performed using a modified technique of that originally described by Billingham, Brent and Medawar.sup.28. Tail skin from donor mice was prepared immediately post-mortem, making a circumferential incision around the base of the tail and then extending the incision distally along the ventral midline. The tail skin was then stripped, placed into cold PBS and fashioned into three 1×1 cm squares.
[0185] Recipient animals were placed under general anesthesia prior to shaving the right flank and preparing skin with chlorhexidine solution. The graft bed was prepared by dissecting skin from the right flank, taking care to preserve underlying subcutaneous adipose tissue (for microvascular blood supply to the graft). Optimally, the skin defect created was slightly larger than the size of the graft (1 mm at each edge), so that the graft remained taught and the risk of seroma formation was minimised. Following placement of the graft onto the graft bed, it was secured in place with methylated flexible collodion (William Ransom & Son Ltd), applied sparingly along the wound edges. The grafts were covered with an iodine-impregnated non-adherent dressing (Inadine®, Johnson and Johnson Medical) and then secured in place with tape. Dressings were removed seven days after skin grafting under a brief general anaesthetic; any animals that managed to remove wound dressings before day 7 were excluded (prospectively) from the experiment as technical failures. Allografts were monitored on a daily basis following the removal of dressings and rejection was defined as more than 90% necrosis by surface area, or when the graft had completely left the recipient. Assessment of graft necrosis was performed in a blinded fashion—after surgery, graft recipients were placed into numbered cages; grafts were monitored according to cage number and were then matched to experimental groups for analysis at the end of each experiment.
[0186] Separately, skin grafts were harvested 7 days after transplantation and specimens were fixed in 10% buffered formalin solution overnight, then stored in 100% ethanol. Specimens were embedded in paraffin and then cut in 4 μm transverse sections. Haematoxylin and eosin (H&E) staining was then performed under automated protocol with a Gemini varistainer (Thermo Scientific), according to the manufacturer's instructions. Histological scoring of rejection was performed in a blinded fashion by a consultant histopathologist. Scoring was performed on three histological sections of each skin graft according to features of vasculitis, folliculitis, dermal inflammation and epidermal degeneration, as described by Zdichavsky et al.sup.29. Images were captured using a Leica DFC290 compound microscope and Leica Application Suite software.
[0187] T cell transfer model of colitis. Foxp3-negative CD4-positive T cells from normal naive Foxp3-GFP C57BL/6 mice were purified by fluorescence cell sorting, and transferred i.v. into naive RAG-1-deficient mice on the C57BL/6 background. Each mouse received 500,000 cells. Groups of recipient mice were implanted with osmotic minipumps (see above) releasing 50 ng per day of Ovalbumin (control protein) or TGM. Minipumps were impanted the day before T cell transfer and continuously released protein for 28 days. At day 60, mice were euthanused, intestinal tissues recovered and stained with haematoxylin and eosin (H&E), and analysed by microscopic evaluation of sections on slides. Colitic inflammation was assessed by a combined histology severity score, comprising a score of 0-3 based on each of the following six parameters: crypt architecture, ulceration, crypt abscesses, goblet cell loss, mucosal inflammatory infiltration and submucosal inflammatory infiltration.
[0188] Statistical Analyses. All statistical analyses were performed using Prism 6.0 (Graphpad Software Inc.). For comparisons of two groups, Student's two-tailed t test was used, assuming unequal variance. When three or more groups were analysed, a one-way ANOVA test was used with Tukey's multiple comparison test. Graft survival curves were compared by Kaplan-Meier analysis; the statistical significance of difference in survival between experimental groups was determined by a log rank chi-square test. P values of <0.05 were considered to be significant; the following symbols were used to indicate significance levels: * denoting p<0.05, ** denoting p<0.01, *** denoting p<0.001 and **** denoting p<0.0001. Sample sizes were chosen empirically on the basis of the lab's previous experience in the calculation of experimental variability (sample sizes for each experiment were not pre-determined by individual power calculations).
[0189] Results
[0190] To identify TGF-β-like activity, the inventors screened H. polygyrus excretory-secretory products (HES) for their ability to activate the MFB-F11 fibroblast cell line in which an alkaline phosphatase reporter is activated by the Smad pathway upon receptor ligation.sup.25. HES proteins were independently fractionated by gel filtration and anion exchange Fast Protein Liquid Chromatography (FPLC), and each fraction assayed for activity on the reporter cell line (
TABLE-US-00007 TABLE 2 Abundance of 18 candidate proteins, including TGM Hp_I03161_IG00349_L1408, in FPLC fractions Gel Filtration Anion Exchange Fraction 9 Fraction 34 Rank Rank Protein emPAI (of 139) emPAI (of 300) GNK0QLK03GIZ2I_L348 0.29 138 0.29 293 Hp_C00269_IG00001_L1007 0.10 87 0.20 213 Hp_C01552_IG00008_L1448 0.21 29 0.06 202 Hp_I03161_IG00349_L1408 0.22 35 0.92 26 Hp_I05436_IG00945_L1999 0.05 130 0.05 246 Hp_I07468_IG01818_L2128 0.53 13 0.74 30 Hp_I10155_IG03162_L783 0.12 61 0.97 39 Hp_I10716_IG03442_L441 0.24 100 0.24 197 Hp_I13201_IG05145_L1858 0.49 11 0.11 140 Hp_I13874_IG05818_L1547 0.13 77 4.33 4 Hp_I14085_IG06029_L1472 0.21 47 0.37 25 Hp_I14567_IG06511_L1339 0.23 44 1.25 26 Hp_I16694_IG08638_L992 0.20 81 0.20 169 Hp_I19747_IG11691_L746 0.12 136 0.12 284 Hp_I24607_IG16551_L570 0.60 49 3.09 20 Hp_I32832_IG24776_L445 0.22 66 0 — Hp_138562_IG30506_L390 0.62 117 1.05 157 Hpb-VAL-20 0.07 110 0 —
[0191] For each candidate, mammalian codon-optimised sequences were synthesized and cloned into the plasmid vector pSecTag2a for transfection of human embryonic kidney HEK293 cells and expression as recombinant proteins with hexa-histidine C-terminal tags. The supernatants of transfected cell cultures were collected and applied directly to the MFB-F11 assay. One transfectant
[0192] (Hp_I03161_IG00349_L1408, the candidate shown in
[0193] The amino acid sequence of active TGM comprises 422 residues, of which the first 18 are predicted to form a classical signal peptide (
[0194] The inventors next tested the ability of purified recombinant TGM protein (
[0195] The MFB-F11 response to HES also exceeded the highest level of the TGF-β-induced signal, but required several log-fold higher concentrations to achieve the same signal as TGM (
[0196] The inventors then established if TGM signalling could be inhibited by antibody to mammalian TGF-β, as could be expected if the parasite molecule interacts with or activates the host molecule in tissue culture medium. MFB-F11 cells were first co-cultured with TGM or hTGF-β1 and 100 μg/ml pan-vertebrate anti-TGF-β antibody (clone 1D11) or MOPC murine IgG control. Anti-TGF-β antibody considerably inhibited the MFB-F11 signal generated from hTGF-β1, but had no impact on TGM (
[0197] To establish if TGM transduces canonical signaling following TGF-β receptor ligation, the MFB-F11 cells were stimulated in the presence or absence of inhibitors of receptor kinase activity. The inventors first tested the kinase inhibitor SB431542 which blocks phosphorylation by TβRI, as well as other TGF-β family type I receptors Alk5 and Alk7.sup.30. SB431542 has previously been found to block the TGF-β-like activity of unfractionated HES, and to render mice more resistant to H. polygyrus infection.sup.9. Both TGM and hTGF-β1 signals were completely ablated in the presence of SB431542 (
[0198] Interestingly, in MFB-F11 cell assays, TGM generally induced a significantly higher signal than hTGF-β1. Further evidence that Hp-TGM can drive an enhanced TGF-β pathway signal was found when stimulating splenocytes from C57BL/6 mice. Following overnight incubation with 20 ng/ml hTGF-β1 or 20 ng/ml Hp-TGM, cells were harvested and assayed for Smad2 phosphorylation by Western blotting. As shown in
[0199] H. polygyrus and HES have previously been shown to induce Foxp3.sup.+ Treg in vitro and in vivo.sup.8,4, 24. The inventors therefore next ascertained if TGM could induce Foxp3.sup.+ Treg differentiation in vitro and if the evidence of enhanced intracellular signalling would be reflected in the level of Foxp3 expression within the induced Treg population. CD4.sup.+CD25.sup.−GFP.sup.−CD62L.sup.hi cells were isolated from Foxp3-GFP reporter mice.sup.30 by MACS and FACS sorting, then cultured for 96 hours in Treg polarising conditions with hTGF-β1 or TGM. TGM was found to effectively induce Foxp3.sup.+ Treg differentiation: at the highest concentration tested (38.1 ng/ml, 0.78 nM) Hp-TGM induced Treg conversion in 90.65% (±3.55%) of all CD4+ cells, compared to 79.65% (±2.55%) induced by a similar molar concentration (10 ng/ml, or 0.78 nM monomeric hTGF-β1) of the human cytokine (
[0200] The inventors then tested whether it could drive expression of Foxp3 in human CD4.sup.+ T cells purified from peripheral blood cultured with anti-CD3/CD28 Dynabeads and variable concentrations of TGM or hTGF-β1 for 96 hrs before assessment of CD25 and Foxp3 expression. As shown in
[0201] To further establish if TGM can induce immune suppressive function in naïve T cells, murine Treg generated from sorted CD4.sup.+CD25.sup.−GFP.sup.−CD62L.sup.hi cells incubated with hTGF-β1 and TGM were added to CD4.sup.+CD25.sup.−GFP.sup.−CD62L.sup.hi responder cells together with soluble anti-CD3 and irradiated APC. Assessment of responder cell proliferation by thymidine incorporation demonstrated that TGM-generated Treg are functionally suppressive in vitro with suppressive capacity equivalent to TGF-β-generated Tregs (
[0202] To test the efficacy of TGM in an in vivo model of immunopathology, the inventors examined its effects in a model of allograft rejection, as helminth parasites and their products have been previously described to prolong the life of tissue transplants.sup.32. The inventors chose the fully-allogeneic skin transplant model, from BALB/c donor mice to C57BL/6 recipients, a system in which median rejection occurs in approximately 9 days, mediated by Th1 and Th17 inflammatory responses. This represents a robust and intense allogeneic reaction.sup.33 but one which is known to be down-modulated by adoptive transfer of Treg populations.sup.34. TGM or HES were administered to mice through osmotic mini-pumps inserted intraperitoneally to continuously release parasite products in a manner akin to live infection. Both TGM and HES conferred a significant extension of allograft life, with median survival extended by ˜5 days relative to untreated controls (
[0203] In parallel experiments, inflammation at the graft site was assessed 7 days post-transplant in animals treated with HES or TGM, in comparison to controls. As shown in
[0204] In allograft recipients exposed to TGM, a significant increase in Foxp3.sup.+ expression was observed in the allograft draining lymph node (
[0205] Mammalian TGF-β is a multi-faceted molecule with effects beyond the regulatory T cell circuit, which can contraindicate its therapeutic use as an immunosuppressant or anti-inflammatory agent. Within the T cell compartment, TGF-β is associated with Th17 differentiation when IL-6 is present in vitro.sup.35,36, or when TGF-β is over-expressed in Freund's complete adjuvant-immunized mice.sup.35; the suppression of ROR-γt expression by TGM in allogeneic skin graft recipients suggests that the parasite ligand does not potentiate Th17 responses in vivo.
[0206] The inventors also demonstrated the protective effects of TGM in a mouse model of colitis. In this model, T cells depleted of the Foxp3-positive regulatory T cell population are transferred into lymphocyte-deficient RAG-1−/− mice; in the absence of regulatory T cells the transferred population become reactive to the intestinal microbiota and recipient mice develop intestinal inflammation known as colitis. TGM was administered through an osmotic minipump implanted subcutaneously on the day prior to T cell transfer, which released 50 ng per day of protein for 28 days. Control mice were implanted with minipumps containing the same quantity of ovalbumin (OVA). As shown in
[0207] Identification of the Functional Domains of TGM from Heligmosomoides polygyrus. Mammalian codon-optimised TGM was synthesized by GENEart and inserted into a holding vector as described in the main manuscript. Amplification and cloning of truncated versions of TGM was then performed by PCR amplification using full length codon-optimised TGM as template DNA and domain-specific primers with restriction sites (Ascl/Apal) and cap sequence placed (gcgcgc) at either end (Table 3).
TABLE-US-00008 TABLE 3 Primer Sequence coTGM_domain1F gcgcgcGGCGCGCCgatgatagcggctgcatg coTGM_domain2F gcgcgcGGCGCGCCagatgcagccccctgccc coTGM_domain3F gcgcgcGGCGCGCCggctgtcctcccctgcctg coTGM_domain4F gcgcgcGGCGCGCCcggtgcaagcctctggaag coTGM_domain5F gcgcgcGGCGCGCCagaggctgcagcagcgacg coTGM_domain1R gcgcgcGGGCCCtctgtcgtcgcattcctgcac coTGM_domain2R gcgcgcGGGCCCgctggcaggacaggtag coTGM_domain3R gcgcgcGGGCCCggggtcggggcacttgcc coTGM_domain4R gcgcgcGGGCCCggcgttgatgcattccatcag coTGM_domain5R gcgcgcGGGCCCcagggtccggatgcc
[0208] PCR reactions to amplify truncated TGM constructs used proofreading Taq polymerase Phusion Hi Fidelity Taq polymerase (NEB) with the following conditions:
[0209] Initial denaturation: 98° C. for 30 s
[0210] 35 cycles of: [0211] Denaturing: 98° C. for 30 s [0212] Annealing: 55-65° C. for 30 s [0213] Extension: 72° C. for 30-90 s (depending on truncation size) [0214] Final extension of 72° C. for 10 mins [0215] 12° C. hold.
[0216] PCR reactions were electrophoresed through a 1.2% agarose gel and a single band of each predicted size of insert was gel extracted and cloned into pSECTag2A (Invitrogen) for transfection and expression in HEK293T cells, as described in the main manuscript.
[0217] The activity of raw expression supernatants were tested for TGF-β activity using the reporter cell line MFB-F11 as described above.
[0218] The full coding sequence of TGM was analyzed and found to be encoded by 11 exons in the parasite genome (
[0219] Further support for a 5-domain model was obtained by searching the in-house transcriptome database for related sequences. Two variants, termed TGM-b and TGM-c, were identified which were missing amino acids 263-343 (the entirety of exons 8+9, domain 4), and amino acids 19-176 (exons 2-5, domains 1+2) (
[0220] Notably TGM-b and TGM-c did not show biological activity. TGM-c is highly divergent (˜50% amino acid difference) while TGMb also had significant amino acid divergence from TGM itself (
[0221] On the strength of these observations, the 5-domain model was tested by expressing a range of truncation constructs as shown in
DISCUSSION
[0222] Helminth parasites are known to exploit the immunoregulatory power of the TGF-β pathway, driving the production of this cytokine by host cells, and promoting longer-term establishment of the parasite in mammalian tissues. Many pathogens, particularly viruses, imitate host cytokines, or even express cytokine genes originally captured from the genome of their host.sup.37. However, no previous example has been reported of a completely unrelated structural product elaborated by parasites that so closely mimics the activity of a crucial host cytokine. Furthermore, the imitation of TGF-β is itself striking, as this mediator is the single most immune suppressive and pro-tolerogenic product of the host immune system involved in a suite of critical immunoregulatory pathways.sup.38. The ability of TGF-β to induce and expand suppressive Treg cells is arguably the most prominent immunological function of TGF-β.sup.12,39, 40, while Treg have been shown to be essential for the survival of several helminth parasites in vivo.sup.4,5 including H. polygyrus.sup.10.
[0223] As metazoa, helminth organisms also encode endogenous members of the TGF-β ligand and receptor families, which in some settings can interact with cognate partners of vertebrate origin.sup.41-44 Further, the inventors had reported that immunomodulatory Treg were induced by H. polygyrus secreted products acting through the TGF-β pathway.sup.9. The present invention now identifies the molecular agent responsible, and shows that rather than belonging to the classical TGF-β family, the parasite molecule represents an unexpected and novel structure. This finding emphasizes the remarkable immunomodulatory strategy of H. polygyrus, which has convergently evolved a unique structure able to signal through the TGF-β pathway and, like TGF-β itself, induce potently suppressive Treg and abate inflammation in vivo.
[0224] Despite signalling through the TGF-β receptors, TβRI and TβRII, and driving Smad phosphorylation, TGM is structurally distinct from the TGF-β molecule. TGM shares no sequence homology with TGF-β, is almost twice the size of a TGF-β homodimer (49 kDa vs. 24 kDa), and is not recognised by pan-vertebrate anti-TGF-β antibodies. Furthermore, TGM is constitutively active, in contrast to the TGF-β ligands which are processed from a longer pre-protein to a mature ˜110-amino acid growth factor domain by proteolytic cleavage at a conserved furin site (RRXR).sup.45.
[0225] TGM is a member of the CCP superfamily defined by a 60-80 amino acid (aa) domain with 4 cysteines and key characteristic residues including a conserved tryptophan. In most eukaryotic species, including nematodes, the protein family has expanded and radiated with extensive diversity of structure and function. Interestingly, searching the genomes of the human hookworms Necator americanus.sup.46 and Ancylostoma duodenale reveals 12-18 members of this gene superfamily, each with low levels of sequence similarity to TGM. It remains to be determined if one or more of these homologues may share the cytokine-like activity of TGM.
[0226] A notable finding has been that TGM stimulates greater expression of Foxp3 compared to that achieved with TGF-β in both murine and human CD4.sup.+ T cells. Intensity of Foxp3 expression by Treg has previously been shown to directly correlate with suppressive ability.sup.47 while high concentrations of TGF-β favour Treg over Th17 differentiation.sup.48. It may be that TGM is able to deliver a stronger signal through the canonical TGF-β receptor cascade, or it may be inured to inhibitory pathways that naturally counteract signals from the mammalian ligand such as the pseudoreceptor BAMBI.sup.49.
[0227] Within the inducible Treg compartment, expression of Foxp3 and consequent regulatory function is in some settings reversible.sup.50,51. As it is possible that TGM drives a more stable and longer-lasting Treg phenotype, and/or one less influenced by inflammatory cytokines such as IL-6, it means new therapies such as autologous transfusion following ex vivo expansion of Treg.sup.52 may be much enhanced in terms of both efficacy and safety.
[0228] The ability of TGM to delay allograft rejection, and to inhibit all three major subsets of effector CD4.sup.+ T cells in vivo, indicates the important therapeutic application of this new molecule. Recombinant TGM offers several advantages including scalable production, a definable mechanism of action and the opportunity for modification to reduce immunogenicity and optimise pharmacokinetic characteristics for pharmacological use. Furthermore, combinations of TGM with currently available immunomodulatory agents will enrich future therapeutic strategies in which the directed manipulation of the different T cell subsets will offer resolution of inflammatory conditions of diverse aetiologies.
TABLE-US-00009 TABLE 1 TGM Assembly Hp_I03161_IG00349_L1408 Nucleotide ACAACCGCATTTATTTCATTCCGCGCTCAGAAAATATCCTTCATAGTGTGCGAATTCCGTCCTTTGTG sequence CACCGATGTACAGTTTTAGTTCCGGGTTCATGCCATTCGCCGTTAGAGCACTCCAGCCGCACCGTTTT ACCTCCACATTCTGCAATCACTTTTGAGCCGGTTGCGTAGAACCTGGCGAAGTCGTCCTTATAACTAT CACTGCCTTCTCCTTTCCTAACAATAACCTTTTCGAAACCAAGTTTATCAAACAAGTCGTCTGAGCTA CAACCTCTTGCGTTAATGCATTCCATAAGCGCAGTAAAATTCCACGTGGCGCCAATGCATTCTCCGA AAATCGGCCCTCCAGTGGAACATGTATACGGCCACTTGCTGCAAACCTTTTTGACGCAGGTGTGTTC AGGGTACTTTCCACTCTTGCCTACTTTCGCGGGTGGGCCTTTCTTTTTGTCTGTCTCGTTGGTCATGGT GAAGTATTCGTAGTGAACAGACTCGTTAGCTTCCAGTGGCTTGCATCTAGGATCTGGGCACTTTCCG ATATTCTTATAGTACTCCCAGTGACTTTCTCCAGTGGTACCACTTTTGTAGCAAATGGCGACAAATTC CCCTGGATCAGCTTCTTGGCTGAGTGCCCTACAACGCCTTCTTGCATGCGTCGGTGAAGGGTAATTT CCAGAACTGTCCTTAGTTACTACTGGTCCGACGGTATGTCGGTCGCCAGCGTATCCGTAGTATTCGT AGAAGACAATTCCATCGTCTGGCAGCGGTGGGCAACCACTGGCTGGGCATGTTGGTGTGCTGGAAA ATTGCCACTCAGCATTGTAGCACATACCGATAATATGACCTTGAACATTGCTGTCTGTAGGAAAGTTT TTGCAGATTCTTTTAATGTATGTTAGTTCAGGATATTTTCCAGAGGCATCTGGATGGACCGTTATATTA AAAATGATTCCTGGATTTACAGTGGCTTTAAGATACTCGAAGCTGACAGTATCGTTAGTCGGCAACG GCGAGCACCTACGATCATCGCATTCTTGTACTCCTTCATAGTAGTACCATTGTGATGCAAGGCAGAT GCCAACGAACCAACCTGTCGTGTCTTCTCCATGCAATCCTTTACAGAACCGTTTAACATGTGTATAAT CAGGATACATTCCAGAATTGTCGATTTGCGCAGGAATTTCTATGTTCTTAGGACCTTTTGCAACGTAT TTGTAGGTGGCAGCTTCATCAGAAAACGGCATGCAGCCGCTGTCATCTGTAGCTGCAACCTCGAGT AGGCCAATCACTACTGTCAGCAGCATTTCAATGTTTTACTAGGAGCAGTTCCATAGGACTTGGCTGA TACAACTCCTGACGGAAGAACTGACCGAAAATCACTCACGCTAACGATAAAGGACCCC Nucleotide ATGCTGCTGACAGTAGTGATTGGCCTACTCGAGGTTGCAGCTACAGATGACAGCGGCTGCATGCCG ORF TTTTCTGATGAAGCTGCCACCTACAAATACGTTGCAAAAGGTCCTAAGAACATAGAAATTCCTGCGC AAATCGACAATTCTGGAATGTATCCTGATTATACACATGTTAAACGGTTCTGTAAAGGATTGCATGG AGAAGACACGACAGGTTGGTTCGTTGGCATCTGCCTTGCATCACAATGGTACTACTATGAAGGAGT ACAAGAATGCGATGATCGTAGGTGCTCGCCGTTGCCGACTAACGATACTGTCAGCTTCGAGTATCTT AAAGCCACTGTAAATCCAGGAATCATTTTTAATATAACGGTCCATCCAGATGCCTCTGGAAAATATC CTGAACTAACATACATTAAAAGAATCTGCAAAAACTTTCCTACAGACAGCAATGTTCAAGGTCATAT TATCGGTATGTGCTACAATGCTGAGTGGCAATTTTCCAGCACACCAACATGCCCAGCCAGTGGTTGC CCACCGCTGCCAGACGATGGAATTGTCTTCTACGAATACTACGGATACGCTGGCGACCGACATACC GTCGGACCAGTAGTAACTAAGGACAGTTCTGGAAATTACCCTTCACCGACGCATGCAAGAAGGCGT TGTAGGGCACTCAGCCAAGAAGCTGATCCAGGGGAATTTGTCGCCATTTGCTACAAAAGTGGTACC ACTGGAGAAAGTCACTGGGAGTACTATAAGAATATCGGAAAGTGCCCAGATCCTAGATGCAAGCCA CTGGAAGCTAACGAGTCTGTTCACTACGAATACTTCACCATGACCAACGAGACAGACAAAAAGAAA GGCCCACCCGCGAAAGTAGGCAAGAGTGGAAAGTACCCTGAACACACCTGCGTCAAAAAGGTTTG CAGCAAGTGGCCGTATACATGTTCCACTGGAGGGCCGATTTTCGGAGAATGCATTGGCGCCACGTG GAATTTTACTGCGCTTATGGAATGCATTAACGCAAGAGGTTGTAGCTCAGACGACTTGTTTGATAAA CTTGGTTTCGAAAAGGTTATTGTTAGGAAAGGAGAAGGCAGTGATAGTTATAAGGACGACTTCGCC AGGTTCTACGCAACCGGCTCAAAAGTGATTGCAGAATGTGGAGGTAAAACGGTGCGGCTGGAGTG CTCTAACGGCGAATGGCATGAACCCGGAACTAAAACTGTACATCGGTGCACAAAGGACGGAATTCG CACACTATGA Amino acid MLLTVVIGLLEVAATDDSGCMPFSDEAATYKYVAKGPKNIEIPAQIDNSGMYPDYTHVKRFCKGLHGEDT Sequence TGWFVGICLASQWYYYEGVQECDDRRCSPLPTNDTVSFEYLKATVNPGIIFNITVHPDASGKYPELTYIKRI CKNFPTDSNVQGHIIGMCYNAEWQFSSTPTCPASGCPPLPDDGIVFYEYYGYAGDRHTVGPVVTKDSSG NYPSPTHARRRCRALSQEADPGEFVAICYKSGTTGESHWEYYKNIGKCPDPRCKPLEANESVHYEYFTMT NETDKKKGPPAKVGKSGKYPEHTCVKKVCSKWPYTCSTGGPIFGECIGATWNFTALMECINARGCSSDD LFDKLGFEKVIVRKGEGSDSYKDDFARFYATGSKVIAECGGKTVRLECSNGEWHEPGTKTVHRCTKDGIRT L* TGM-a Assembly Hp_I03162_IG00349_L1408 Nucleotide ACAACCACATTTATTTCATTCCAAGATAAGAGAATATCCTTCATAGTGCCTAATTCCTTCGCTTGTGC sequence ACCGATGCACAGTTCTTGTTTCCGAATCATGCCATTCGCCGTTAGAGCACTCCAGTCGCACCGTTTTA CCTTTACATTCTGCATTCACTTTCGAACCGGTTGTGTAGAACCTGACGTAGTCGTCTTTATAACTATCG CTGCCTTCTCCTTCCCTAACCATAACTATTTCAAAGCCCAATTTATTAAACAAGTCGCCTCCGTCACA ACCCCTTGCGTTTAAGCATTCGTCAAGTGCAGTAAAATTCCACTGGCCGTCAAGGCATTCTCCGAAA ATCGGCCCTTTAACCGAACATGTATACGGTGACTTGTCGCAAAATTTTCTGACACACGTGTGTTGAG AGTATTTTCCACCCTTATCTACTTGCGCGGGCGTGCCTTCCTTTTTGCCTGTCTCGTTGGCCATGGTGA AGTATTCGTAGCGAACAGACTCGTCAGCTTTCAGTGGCTTGCATCTAGGATCTGGGCAATTCTTTATA TACTTATAGTACTGCCAGTGACTTTCTCCAGTGGTACAACTTTTGTAGCAAATGGCGACAAATTCCCC TGTATCAGCTTTTTGGCTGAGTGCCCTACAACGCCTTCTTGCATGCGTCGGTGAAGGGTAATTTCCAG AACTGTCCTTAGTTACTACTGGTCCGACGGTATGTCGGTCGCCAGCGTATCCGTAGTATTCGTAGAA GACAATTCCATCGTCTGGCAGCGGTGGGCAACCACTGGCTGGGCATGTTGGGTGTGCTGGAAAATT GCCACTCAGCATTGTAGCACATACCGATAATATGACCTTGAACATTGCTGTCTGTAGGAAAGTTTTTG CAGATTCTTTTAATGTATGTTAGTTCAGGATATTTTCCAGAGGCATCTGGATGGACCGTTATATTAAA AATGATTCCTGGATTTACAGTGGCTTTAAGATACTCGAAGCTGACAGTATCGTTAGTCGGCAACGGC GAGCACCTACGATCATCGCATTCTTGTACTCCTTCATAGTAGTACCATTGTGATGCAAGGCAGATGC CAACGAACCAACCTGTCGTGTCTTCTCCATGCAATCCTTTACAGAACCGTTTAACATGTGTATAATCA GGATACATTCCAGAATTGTCGATTTGCGCAGGAATTTCTATGTTCTTAGGACCTTTTGCAACGTATTT GTAGGTGGCAGCTTCATCAGAAAACGGCATGCAGCCGCTGTCATCTGTAGCTGCAACCTCGAGTAG GCCAATCACTACTGTCAGCAGCATTTCAATGTTTTACTAGGAGCAGTTCCATAGGACTTGGCTGATA CAACTCCTGACGGAAGAACTGACCGAAAATCACTCACGCTAACGATAAAGGACCCC Nucleotide ATGCTGCTGACAGTAGTGATTGGCCTACTCGAGGTTGCAGCTACAGATGACAGCGGCTGCATGCCG sequence TTTTCTGATGAAGCTGCCACCTACAAATACGTTGCAAAAGGTCCTAAGAACATAGAAATTCCTGCGC ORF AAATCGACAATTCTGGAATGTATCCTGATTATACACATGTTAAACGGTTCTGTAAAGGATTGCATGG AGAAGACACGACAGGTTGGTTCGTTGGCATCTGCCTTGCATCACAATGGTACTACTATGAAGGAGT ACAAGAATGCGATGATCGTAGGTGCTCGCCGTTGCCGACTAACGATACTGTCAGCTTCGAGTATCTT AAAGCCACTGTAAATCCAGGAATCATTTTTAATATAACGGTCCATCCAGATGCCTCTGGAAAATATC CTGAACTAACATACATTAAAAGAATCTGCAAAAACTTTCCTACAGACAGCAATGTTCAAGGTCATAT TATCGGTATGTGCTACAATGCTGAGTGGCAATTTTCCAGCACACCCAACATGCCCAGCCAGTGGTTG CCCACCGCTGCCAGACGATGGAATTGTCTTCTACGAATACTACGGATACGCTGGCGACCGACATAC CGTCGGACCAGTAGTAACTAAGGACAGTTCTGGAAATTACCCTTCACCGACGCATGCAAGAAGGCG TTGTAGGGCACTCAGCCAAAAAGCTGATACAGGGGAATTTGTCGCCATTTGCTACAAAAGTTGTACC ACTGGAGAAAGTCACTGGCAGTACTATAAGTATATAAAGAATTGCCCAGATCCTAGATGCAAGCCA CTGAAAGCTGACGAGTCTGTTCGCTACGAATACTTCACCATGGCCAACGAGACAGGCAAAAAGGAA GGCACGCCCGCGCAAGTAGATAAGGGTGGAAAATACTCTCAACACACGTGTGTCAGAAAATTTTGC GACAAGTCACCGTATACATGTTCGGTTAAAGGGCCGATTTTCGGAGAATGCCTTGACGGCCAGTGG AATTTTACTGCACTTGACGAATGCTTAAACGCAAGGGGTTGTGACGGAGGCGACTTGTTTAATAAAT TGGGCTTTGAAATAGTTATGGTTAGGGAAGGAGAAGGCAGCGATAGTTATAAAGACGACTACGTCA GGTTCTACACAACCGGTTCGAAAGTGAATGCAGAATGTAAAGGTAAAACGGTGCGACTGGAGTGCT CTAACGGCGAATGGCATGATTCGGAAACAAGAACTGTGCATCGGTGCACAAGCGAAGGAATTAGG CACTATGAAGGATATTCTCTTATCTTGGAATGA Amino acid MLLTVVIGLLEVAATDDSGCMPFSDEAATYKYVAKGPKNIEIPAQIDNSGMYPDYTHVKRFCKGLHGEDT sequence TGWFVGICLASQWYYYEGVQECDDRRCSPLPTNDTVSFEYLKATVNPGIIFNITVHPDASGKYPELTYIKRI CKNFPTDSNVQGHIIGMCYNAEWQFSSTPTCPASGCPPLPDDGIVFYEYYGYAGDRHTVGPVVTKDSSG NYPSPTHARRRCRALSQKADTGEFVAICYKSCTTGESHWQYYKYIKNCPDPRCKPLKADESVRYEYFTMA NETGKKEGTPAQVDKGGKYSQHTCVRKFCDKSPYTCSVKGPIFGECLDGQWNFTALDECLNARGCDGG DLFNKLGFEIVMVREGEGSDSYKDDYVRFYTTGSKVNAECKGKTVRLECSNGEWHDSETRTVHRCTSEGI RHYEGYSLILE* Comments identical N-terminal domains (1-228), 49 changes 229-422, 8 additional aa TGM-b Assembly Hp_I03163_IG00349_L1163 Nucleotide AACCACATTTATTTCACTCCAAGATGAGAAAATATTCTTCATGGTGCGCGAAGTCCTTCCTTTGTGCA Sequence CCGGTGCACAGTTTTAGTTCCGGGATCATGCCATTCACCGTTAGAGCACTCCAGCCGCACCGTGTTA CCTTTACATTCGGCATTCACTTTCGAACCGGTTGCGTAGAACCGGGCAAAGTCGTCCTTATAACTATC ACTGCCTTCTTCTTCTCTAACCATAACTCCTTCAAAGCCCAGTTTATCAAACAAGTCGTCACTGTTACA ACCCCTAGGATCTGGGCACTTCCTTATATGACTGTAGTAATCCCAATGACTTTCGCCAGTGGTACCA CATCTGTAGCAAATGCCAACAAATTCCCCTGGATCAGCTTTTTGGCTGAGTGCCCTACAACGCCTTCT TGCATGCGTCGGTGAAGGGTAATTTCCAGAACTGTCCTTAGTTACTGCTGGTCCGACGGTATGTCGA TCGCCAGCGTATCCGTAGTATTCGTGGAAGACGATTCCATCGTCTGGCAGCGGTGGGCAACCAATT GAAGGGCTTGTTGGTGTGCTGGAAAACCGCCATTCAGCATTGTAGCACATGCCGATAATATGACCTT GAACTTTGCTGTCAGTAGGAAAATTTTTGCAGATTCTTTTAATGTATGTTAGTTCAGGATATTTTCCAG TGGCATCTGGATGGACCGTTATATTAAAAATGATTCCTGGATTTACAGTGGCTTTAAGATACTCGAA GGTGACAGTATCGTCCGTCGACAACGGCAAGCACCCACGACCTCCGCATTCTTGTACTCCTTGATAG TAGACCCATTCTGATCCAGGGCCTATGCCAACGAACCAACCTGTCGTGTCTTCTCCATGCAATCCTTT ACAAAACCGTTTAACATGTGTATAATCAGGATACATTCCAGAATTGTCGATTTGCGCAGGAATTTCTA TGTTCTTAGGACCTTTTGCAACGTATTTGTAGGTGGCAGCTTCATCAGAAAACGGCATGCAGCCGCT GTCATCTGTAGCTGCAACCTCGAGTAGGCCAATCACTACTGTCAGCAGCATTTCAATGTTTTACTAG GAGCAGTTCCATAGGACTTGGCTGATACAACTCCTGACGGAAGAACTGACCGAAAATCACTCACGC TAACGATAAAGGACCCC Nucleotide ATGCTGCTGACAGTAGTGATTGGCCTACTCGAGGTTGCAGCTACAGATGACAGCGGCTGCATGCCG Sequence TTTTCTGATGAAGCTGCCACCTACAAATACGTTGCAAAAGGTCCTAAGAACATAGAAATTCCTGCGC ORF AAATCGACAATTCTGGAATGTATCCTGATTATACACATGTTAAACGGTTTTGTAAAGGATTGCATGGA GAAGACACGACAGGTTGGTTCGTTGGCATAGGCCCTGGATCAGAATGGGTCTACTATCAAGGAGTA CAAGAATGCGGAGGTCGTGGGTGCTTGCCGTTGTCGACGGACGATACTGTCACCTTCGAGTATCTTA AAGCCACTGTAAATCCAGGAATCATTTTTAATATAACGGTCCATCCAGATGCCACTGGAAAATATCC TGAACTAACATACATTAAAAGAATCTGCAAAAATTTTCCTACTGACAGCAAAGTTCAAGGTCATATT ATCGGCATGTGCTACAATGCTGAATGGCGGTTTTCCAGCACACCAACAAGCCCTTCAATTGGTTGCC CACCGCTGCCAGACGATGGAATCGTCTTCCACGAATACTACGGATACGCTGGCGATCGACATACCG TCGGACCAGCAGTAACTAAGGACAGTTCTGGAAATTACCCTTCACCGACGCATGCAAGAAGGCGTT GTAGGGCACTCAGCCAAAAAGCTGATCCAGGGGAATTTGTTGGCATTTGCTACAGATGTGGTACCA CTGGCGAAAGTCATTGGGATTACTACAGTCATATAAGGAAGTGCCCAGATCCTAGGGGTTGTAACA GTGACGACTTGTTTGATAAACTGGGCTTTGAAGGAGTTATGGTTAGAGAAGAAGAAGGCAGTGATA GTTATAAGGACGACTTTGCCCGGTTCTACGCAACCGGTTCGAAAGTGAATGCCGAATGTAAAGGTA ACACGGTGCGGCTGGAGTGCTCTAACGGTGAATGGCATGATCCCGGAACTAAAACTGTGCACCGGT GCACAAAGGAAGGACTTCGCGCACCATGA Amino acid MLLTVVIGLLEVAATDDSGCMPFSDEAATYKYVAKGPKNIEIPAQIDNSGMYPDYTHVKRFCKGLHGEDT Sequence TGWFVGIGPGSEWVYYQGVQECGGRGCLPLSTDDTVTFEYLKATVNPGIIFNITVHPDATGKYPELTYIKRI CKNFPTDSKVQGHIIGMCYNAEWRFSSTPTSPSIGCPPLPDDGIVFHEYYGYAGDRHTVGPAVTKDSSG NYPSPTHARRRCRALSQKADPGEFVGICYRCGTTGESHWDYYSHIRKCPDPRGCNSDDLFDKLGFEGV MVREEEGSDSYKDDFARFYATGSKVNAECKGNTVRLECSNGEWHDPGTKTVHRCTKEGLRAP* Comments 21 changes in N terminus (21/228 = 90.8% identity). Missing Cys-79. Missing exons 8 and 9 (aa 263-343); between 343-422, 13 aa changes (85.6% identity) TGM-c Assembly Hp_I18045_IG09989_L856 Nucleotide TGGTTTAATTACCCAAGTTTTGAGGGGATCATTGCGGAACTCGACGCTCCACCAAGATGCTATCATT Sequence ATTCATTGCCATCGGATTGCTTGAGGCTGCTGGATCAAGTTGTCCACCCGTGGGCGATGCAGCAATT AAGGACAGTCTCGAAAATTATCCTCCGAATACGCATGCAAGAAGACATTGCAAAGCACTCAGCAAA AAAGCTGACCCAGGAGAATTTGTCGCCATTTGCTACCAAAGAAGAGGCACTAGTGAAAGTCAATGG CAGTATTATCCTAGAATAGCATCATGTCCAGACCCTAGGTGCAAGCCTCTCGAAAAAAACGATTCTG TTAGCTATGAATATTTCACAAAACCCACTAAAGGACTGAAGATGGGTTCAATCACAAAGCCGGACA AAAGTGGAAAGTACCCTGAAGAAACTTTTGTTAGAAGGTATTGCAATGACCTTCCAAGGAACAGCC TAGCGCAAGGAAAAACCTACGCAGAGTGCTTGGATTCAGAGTGGAAGCTTAAAAATTTGCCAGACT GTCGATTCGCAGCAGGATGTGACGAAGAATATCTGCTTGAGAAGTTAATGTTCGTTGATATTTCGTA CTGGGGAAAAGATGCAGCGAAATTTTCCGATGACAAAACATATAGGTATTATCGGCCCGGTTCGAA AGTTACTGCGAAGTGCAAAGGTAAATCTGTGAAGTTAACATGTGTTGACGGCGGCTACTGGGTTAC AGTGGACGGCAGAAAGGCGCTCTGCACATGAGACGGTCCTCACAGTTGAAGTGCAATAAATATTTT CGCTACCAGATGATGAAACTGTCTGGTACGAATACTACGGATACGTTGACGGTCGACATA Nucleotide ATGCTATCATTATTCATTGCCATCGGATTGCTTGAGGCTGCTGGATCAAGTTGTCCACCGCTACCAGA Sequence TGATGAAACTGTCTGGTACGAATACTACGGATACGTTGACGGTCGACATACCGTGGGCGATGCAGC ORF AATTAAGGACAGTCTCGAAAATTATCCTCCGAATACGCATGCAAGAAGACATTGCAAAGCACTCAG CAAAAAAGCTGACCCAGGAGAATTTGTCGCCATTTGCTACCAAAGAAGAGGCACTAGTGAAAGTCA ATGGCAGTATTATCCTAGAATAGCATCATGTCCAGACCCTAGGTGCAAGCCTCTCGAAAAAAACGAT TCTGTTAGCTATGAATATTTCACAAAACCCACTAAAGGACTGAAGATGGGTTCAATCACAAAGCCGG ACAAAAGTGGAAAGTACCCTGAAGAAACTTTTGTTAGAAGGTATTGCAATGACCTTCCAAGGAACA GCCTAGCGCAAGGAAAAACCTACGCAGAGTGCTTGGATTCAGAGTGGAAGCTTAAAAATTTGCCAG ACTGTCGATTCGCAGCAGGATGTGACGAAGAATATCTGCTTGAGAAGTTAATGTTCGTTGATATTTC GTACTGGGGAAAAGATGCAGCGAAATTTTCCGATGACAAAACATATAGGTATTATCGGCCCGGTTC GAAAGTTACTGCGAAGTGCAAAGGTAAATCTGTGAAGTTAACATGTGTTGACGGCGGCTACTGGGT TACAGTGGACGGCAGAAAGGCGCTCTGCACATGA Amino acid MLSLFIAIGLLEAAGSSCPPLPDDETVWYEYYGYVDGRHTVGDAAIKDSLENYPPNTHARRHCKALSKKA Sequence DPGEFVAICYQRRGTSESQWQYYPRIASCPDPRCKPLEKNDSVSYEYFTKPTKGLKMGSITKPDKSGKYPE ETFVRRYCNDLPRNSLAQGKTYAECLDSEWKLKNLPDCRFAAGCDEEYLLEKLMFVDISYWGKDAAKFS DDKTYRYYRPGSKVTAKCKGKSVKLTCVDGGYWVTVDGRKALCT Comments Missing exons 2, 3, 4 and 5. Only 254 aa, over which shows 117 (46.0%) identity TGM-d Assembly Hp_I03160_IG00349_L1402 Nucleotide AACCACATTTATTTCACTCCAAGATGAGAAAATATTCTTCATGGTGCGCGAAGTCCTTCCTTTGTGCA Sequence CGATGCACAGTTTTAGCTCCCAAATCATGCCATTCGCCGTTAGAGCACTCCAGCCGCGCCGTTTCAC CTCGACATTGTGCACTGACTTTAGAACCGGTTGCGTAGAACAAGACGTAGGCGTCCCTATAACTATC ACTGAATTCTCCTTCCCTAACCATTATTTCTTTGAAGCCCAGCTCAAACAAGTCGTTTTCGTCACAAC CTCTTGCGTTAATGCATTCCATAAGCGCAGTAAAATTCCACGTGGCGCCAATGCATTCTCCGAAAAT CGGCCCTCCAGCGGAACATGTATACGGCCACTTGCTGCAAACCTTTTTGACGCAGGTGTGTTCAGG GTACTTTCCACTCTTGCCTACTTTCGCGGGTGGGCCTTTCTTTTTGTCTGTCTCGTTGGTCATGGTGAA GTATTCGTAGTGAACAGACTCGTTAGCTTCCAGTGGCTTGCATCTAGGATCTGGGCACTTTCCGATAT TCTTATAGTACTCCCAGTGACTTTCTCCAGTGGTACCACTTTTGTAGCAAATGGCGACAAATTCCCCT GGATCAGCTTCTTGGCTGAGTGCCCTACAACGCCTTCTTGCATGCGTCGGTGAAGGGTAATTTCCAG AACTGTCCTTAGTTACTACTGGTCCGACGGTATGTCGGTCGCCAGCGTATCCGTAGTATTCGTAGAA GACAATTCCATCGTCTGGCAGCGGTGGGCAACCACTGGCTGGGCATGTTGGTGTGCTGGAAAATTG CCACTCAGCATTGTAGCACATACCGATAATATGACCTTGAACATTGCTGTCTGTAGGAAAGTTTTTGC AGATTCTTTTAATGTATGTTAGTTCAGGATATTTTCCAGAGGCATCTGGATGGACCGTTATATTAAAA ATGATTCCTGGATTTACAGTGGCTTTAAGATACTCGAAGCTGACAGTATCGTTAGTCGGCAACGGCG AGCACCTACGATCATCGCATTCTTGTACTCCTTCATAGTAGTACCATTGTGATGCAAGGCAGATGCC AACGAACCAACCTGTCGTGTCTTCTCCATGCAATCCTTTACAGAACCGTTTAACATGTGTATAATCAG GATACATTCCAGAATTGTCGATTTGCGCAGGAATTTCTATGTTCTTAGGACCTTTTGCAACGTATTTGT AGGTGGCAGCTTCATCAGAAAACGGCATGCAGCCGCTGTCATCTGTAGCTGCAACCTCGAGTAGGC CAATCACTACTGTCAGCAGCATTTCAATGTTTTACTAGGAGCAGTTCCATAGGACTTGGCTGATACA ACTCCTGACGGAAGAACTGACCGAAAATCACTCACGCTAACGATAAAGGACCCC Nucleotide ATGCTGCTGACAGTAGTGATTGGCCTACTCGAGGTTGCAGCTACAGATGACAGCGGCTGCATGCCG Sequence TTTTCTGATGAAGCTGCCACCTACAAATACGTTGCAAAAGGTCCTAAGAACATAGAAATTCCTGCGC ORF AAATCGACAATTCTGGAATGTATCCTGATTATACACATGTTAAACGGTTCTGTAAAGGATTGCATGG AGAAGACACGACAGGTTGGTTCGTTGGCATCTGCCTTGCATCACAATGGTACTACTATGAAGGAGT ACAAGAATGCGATGATCGTAGGTGCTCGCCGTTGCCGACTAACGATACTGTCAGCTTCGAGTATCTT AAAGCCACTGTAAATCCAGGAATCATTTTTAATATAACGGTCCATCCAGATGCCTCTGGAAAATATC CTGAACTAACATACATTAAAAGAATCTGCAAAAACTTTCCTACAGACAGCAATGTTCAAGGTCATAT TATCGGTATGTGCTACAATGCTGAGTGGCAATTTTCCAGCACACCAACATGCCCAGCCAGTGGTTGC CCACCGCTGCCAGACGATGGAATTGTCTTCTACGAATACTACGGATACGCTGGCGACCGACATACC GTCGGACCAGTAGTAACTAAGGACAGTTCTGGAAATTACCCTTCACCGACGCATGCAAGAAGGCGT TGTAGGGCACTCAGCCAAGAAGCTGATCCAGGGGAATTTGTCGCCATTTGCTACAAAAGTGGTACC ACTGGAGAAAGTCACTGGGAGTACTATAAGAATATCGGAAAGTGCCCAGATCCTAGATGCAAGCCA CTGGAAGCTAACGAGTCTGTTCACTACGAATACTTCACCATGACCAACGAGACAGACAAAAAGAAA GGCCCACCCGCGAAAGTAGGCAAGAGTGGAAAGTACCCTGAACACACCTGCGTCAAAAAGGTTTG CAGCAAGTGGCCGTATACATGTTCCGCTGGAGGGCCGATTTTCGGAGAATGCATTGGCGCCACGTG GAATTTTACTGCGCTTATGGAATGCATTAACGCAAGAGGTTGTGACGAAAACGACTTGTTTGAGCTG GGCTTCAAAGAAATAATGGTTAGGGAAGGAGAATTCAGTGATAGTTATAGGGACGCCTACGTCTTG TTCTACGCAACCGGTTCTAAAGTCAGTGCACAATGTCGAGGTGAAACGGCGCGGCTGGAGTGCTCT AACGGCGAATGGCATGATTTGGGAGCTAAAACTGTGCATCGTGCACAAAGGAAGGACTTCGCGCA CCATGAAGAATATTTTCTCATCTTGGAGTGA Amino acid MLLTVVIGLLEVAATDDSGCMPFSDEAATYKYVAKGPKNIEIPAQIDNSGMYPDYTHVKRFCKGLHGEDT Sequence TGWFVGICLASQWYYYEGVQECDDRRCSPLPTNDTVSFEYLKATVNPGIIFNITVHPDASGKYPELTYIKRI CKNFPTDSNVQGHIIGMCYNAEWQFSSTPTCPASGCPPLPDDGIVFYEYYGYAGDRHTVGPVVTKDSSG NYPSPTHARRRCRALSQEADPGEFVAICYKSGTTGESHWEYYKNIGKCPDPRCKPLEANESVHYEYFTMT NETDKKKGPPAKVGKSGKYPEHTCVKKVCSKWPYTCSAGGPIFGECIGATWNFTALMECINARGCDEND LFELGFKEIMVREGEFSDSYRDAYVLFYATGSKVSAQCRGETARLECSNGEWHDLGAKTVHRAQRKDFA HHEEYFLILE* Comments identical 1-318; 34 changes 319-422 (67.3% identity), 8 additional aa TGM-e Nucleotide ACAACCACATTTATTTCATTCCAAGCTGATGAAATATCCTTCATAGTGCGCGGATTCCTTCC Sequence TTTGTGCACCGATGCACAGTTTTAGTTCCGGGATCATGCCATTCACCGTCAGAGCACTCCAGCTGCA CCGTTTTACCTTTACATTCCGCATTCACTTTCGAACCGGTTGCGTAGAACCTGACGAAGTCGTCCTTA TAACTATCACTGCCTTCTTCTTCTCTAACCATAACTCCTTCAAAGCCCAGTTTATCAAACAAGTCGTCA CTGTTGCAACCCCTTGCGTTTAAGCATTCGTCAAGTGCAGTAAAATTCCACTGGCCGTCAAGGCATT CTCCGAAAATCGGCCCTTTAACCGAACATGTATACGGTGACTTGTCGCAAAATTTTCTGACACACGT GTGTTGAGGGTACTTTCCACCCTTGTCTACTTCCGCGGGCGTGCCTTCCTTTCTGCCCGTCTCGTTGG TCATGGTGAAGTATTCGTAGTGAACAGACACGTTAGTTTCCAGTGGCTTGCATCTAGGATCTGGGCA TTTCCTTATATGACTGTAGTAATCCCAATGACTTTCGCCAGTCGTACCGCTTTTGTAGCAAATGCCAA CAAATTCTCCTGGATCAGCTTTTTGGCTGAGTGCCCTACAACGCCTTCTTGCATGCGTTTGTGGAGGG TAATTTCCAGAACTGTCCTTAGATACTGCTCGTCCGACGGTATGTCGATTGCCAGCGTATCCGTAGTA TTCGTAGAAGACAATTCCATCATCTGGTAGCGGTGGGCAACCACTGGGTGGGCATGTTGGTGTGCT AGAAAACCGCCATTCAGCATTGTAGCACATGCCGATAATATGGCCTTGAACTTTGCTGTCAGCAGGA AAATTTTTGCAGATTCTTTTAATGTATGTTAGTTCAGGATATTTTCCAGAAGCATCTGGATGAACCGTT ATATTAAAATTGATTCCTGCATTTACTGTGGCTTTAAGATACTCGTAGGTGACAGTATCGTTCGTCGG CAACGGCGAGCACCTACGGTCTTGGCATTCTTGTACTCCTTGATAGTAGACCCATTCTGATCCAAGG CAAATGCCGACGTACCTACCTGTCTTTTCTTCTCCATGCAATCCTTTACAAAACCGTTTAACATGTGTA TGATCAGGATACGCTCCAGAGCTGTCATTTTGTGCAGGAGTTTCGTCGTTTCTAGAACGTTCTGTTAA ATATTTATAGGAGGCGGTTTCATCAGAAAATGGCATACAGCCGCTGGCGTCTGTAGCTGCAACCTCG AGTAGGCCAATTAATACAATCAGCAGCATTTCATCACGCTAACGATAAAGGGCCGTTCC Nucleotide ATGCTGCTGATTGTATTAATTGGCCTACTCGAGGTTGCAGCTACAGACGCCAGCGGCTGTATGCCAT Sequence TTTCTGATGAAACCGCCTCCTATAAATATTTAACAGAACGTTCTAGAAACGACGAAACTCCTGCACA ORF AAATGACAGCTCTGGAGCGTATCCTGATCATACACATGTTAAACGGTTTTGTAAAGGATTGCATGGA GAAGAAAAGACAGGTAGGTACGTCGGCATTTGCCTTGGATCAGAATGGGTCTACTATCAAGGAGTA CAAGAATGCCAAGACCGTAGGTGCTCGCCGTTGCCGACGAACGATACTGTCACCTACGAGTATCTT AAAGCCACAGTAAATGCAGGAATCAATTTTAATATAACGGTTCATCCAGATGCTTCTGGAAAATATC CTGAACTAACATACATTAAAAGAATCTGCAAAAATTTTCCTGCTGACAGCAAAGTTCAAGGCCATAT TATCGGCATGTGCTACAATGCTGAATGGCGGTTTTCTAGCACACCAACATGCCCACCCAGTGGTTGC CCACCGCTACCAGATGATGGAATTGTCTTCTACGAATACTACGGATACGCTGGCAATCGACATACCG TCGGACGAGCAGTATCTAAGGACAGTTCTGGAAATTACCCTCCACAAACGCATGCAAGAAGGCGTT GTAGGGCACTCAGCCAAAAAGCTGATCCAGGAGAATTTGTTGGCATTTGCTACAAAAGCGGTACGA CTGGCGAAAGTCATTGGGATTACTACAGTCATATAAGGAAATGCCCAGATCCTAGATGCAAGCCAC TGGAAACTAACGTGTCTGTTCACTACGAATACTTCACCATGACCAACGAGACGGGCAGAAAGGAAG GCACGCCCGCGGAAGTAGACAAGGGTGGAAAGTACCCTCAACACACGTGTGTCAGAAAATTTTGC GACAAGTCACCGTATACATGTTCGGTTAAAGGGCCGATTTTCGGAGAATGCCTTGACGGCCAGTGG AATTTTACTGCACTTGACGAATGCTTAAACGCAAGGGGTTGCAACAGTGACGACTTGTTTGATAAAC TGGGCTTTGAAGGAGTTATGGTTAGAGAAGAAGAAGGCAGTGATAGTTATAAGGACGACTTCGTCA GGTTCTACGCAACCGGTTCGAAAGTGAATGCGGAATGTAAAGGTAAAACGGTGCAGCTGGAGTGCT CTGACGGTGAATGGCATGATCCCGGAACTAAAACTGTGCATCGGTGCACAAAGGAAGGAATCCGC GCACTATGA Amino acid MLLIVLIGLLEVAATDASGCMPFSDETASYKYLTERSRNDETPAQNDSSGAYPDHTHVKRFCKGLHGEEK Sequence TGRYVGICLGSEWVYYQGVQECQDRRCSPLPTNDTVTYEYLKATVNAGINFNITVHPDASGKYPELTYIK RICKNFPADSKVQGHIIGMCYNAEWRFSSTPTCPPSGCPPLPDDGIVFYEYYGYAGNRHTVGRAVSKDSS GNYPPQTHARRRCRALSQKADPGEFVGICYKSGTTGESHWDYYSHIRKCPDPRCKPLETNVSVHYEYFT MTNETGRKEGTPAEVDKGGKYPQHTCVRKFCDKSPYTCSVKGPIFGECLDGQWNFTALDECLNARGCN SDDLFDKLGFEGVMVREEEGSDSYKDDFVRFYATGSKVNAECKGKTVQLECSDGEWHDPGTKTVHRCT KEGIRAL Notes 78 changes throughout, same length (81.5% identity) TGM-f Assembly Hp_I03144_IG00345_L1870 Nucleotide TTTTGTCCAAAGAATCGTTTATTCAACTGTGGCAAACACTGAGTTTATTCGTCATAGGTGACTCCGCT Sequence ATCAGTGCATCGTATTTTAGCGAATTTTCTCTCATTCTCGGCATGACGACGTCCTTGCCATCCACCTTC AAAGCATTCAAGATCTGCCGGGTTACCTTTGCAAAGTGCTTGTATTTTCGAACCAGGTCCGTAAAAT GTAGAAACAGTATCAGGTTTCTTGTATACTGCCACCGCAAGACGTGTATCGACTATAGTTGAGGTGA ATTTCAGATCCAAATAAACCTCGTTTATATTGCAGCTCCCCTGTGGTGGACATGGCCATGTGTTGCG GTGCACCCATCCGTTCGCGCCGCATTTGGAGATATCCCCTTGAGCCCTGCTGTTTACGTCTAGTTTCT TGCAAATCCTCCGAGCATACGAGCCCACAGCGAATTTCCCACTCCAGTCTAGTACGGCAATATTTGC GAAATCGTTACTTGTTTGCTCACTTTTAAAGTACTTGTATTCCACGGTGTCGTTCTCGGCCAACGGTA AGCATCCAAGAATAGGACATTGTAGCACCGTCGCTTCATTCTCCTTCACCCATTCGCCTTTGCTGCAT CTTCCAGCGATTTCTCCTAAGTTTTTGTTATCGCCAGTTGCCTTCTTACAAAACATCTTTGCAGACGTG CGTTCGGGGTATTTTCCAGACCGTGGCTTTACAGTGGAGGTGCTTAGTGTATAGTAGCCTGTGCGTG ATATATTGAGATACTCATATCTTACGGTGTCGTTGTCCGTAAGAGGGGCGCATTCACGCCCAGGGCA TTCCGGTATGATTTGGTACTTTTCTGGCTTCCATTGGCGATTGCTGCATTGTGCAATAATTGTCCCTGT TTTTTCGCCTATTCTAGTCGGTATCTTACAGATGACTATCGCAAACGTGCCGTCAGGGTATTCTCCGC TCTGCGGTGTCGCTTCCTTTGCGACGACAGTTTGGTCAGGAGACATTGTGTATTGGAGATACTTATAC GTAACGGTACTGTTGGCTTTATATGTTATGGTACAGCCAGGAACTGGGCACTGTTTTATATTATGATA GTACACCCACCGACTTTCACCAGTATCACTTCTTTCGTGGCAAATGGCGACAAGTTCCCCTTGATCA ACCTTTACGCTCCGTGCTTGGCAATGCCTTCTTGCATGCGTCTGTGGAGGGTAATTTCCAGAACTGTC CTTAGTTACTGCTTGTCCGACGACATCTCGACTCCCAGCGTATTCATAGTATTCGTGCCGGAATTTGT CATTGTCTTGTATCGGTGGGCAACCATAGGGTGGGCATACTGGTGCGCTGGAGAACCTCCATTCAG CATTGTAGCACATGCCGACAATAACACCTCGAACTTTGCTGTTTGCAGGAAAATTTTTGCATATTCTT CTTATGTATGTTAGTTCAGGATATTTTCCGCTGTCATCTGGATTGGCACTTGTATCATAACTGATTCTC GAAGAATTTAGAGTAGCTTTACGATACTCGTAGCTGACAGTATCGCTCTCAGACAACGGCGAGCATC TACGGTCTCGGCATTCTTGTACTCCCATATAATAGACCCATTCCGATCGATGGCACAGGCCGATGAA CTCACCTGTCTTATCTTCTCCATGCAATCCTTTACAAAACCGTTTCACGTGCGTATACTCAGGATACAT TCCAGAACTGTCCTTTCGCGCAGGTGTTTCCTCTTTATTAGAAGTTTGTGCAAAATATTGGTAGGTGT CAGTTTCTTCAGATAATGGCATGCAGCTGTTGTCGCCTGCAGCCGAAGCCTCGAGAAGGCCAATTAC TACAATCAGCAGCATTTCACTCAAACTTGGGTAATTAAACC Nucleotide ATGCTGCTGATTGTAGTAATTGGCCTTCTCGAGGCTTCGGCTGCAGGCGACAACAGCTGCATGCCAT Sequence TATCTGAAGAAACTGACACCTACCAATATTTTGCACAAACTTCTAATAAAGAGGAAACACCTGCGCG ORF AAAGGACAGTTCTGGAATGTATCCTGAGTATACGCACGTGAAACGGTTTTGTAAAGGATTGCATGG AGAAGATAAGACAGGTGAGTTCATCGGCCTGTGCCATCGATCGGAATGGGTCTATTATATGGGAGT ACAAGAATGCCGAGACCGTAGATGCTCGCCGTTGTCTGAGAGCGATACTGTCAGCTACGAGTATCG TAAAGCTACTCTAAATTCTTCGAGAATCAGTTATGATACAAGTGCCAATCCAGATGACAGCGGAAAA TATCCTGAACTAACATACATAAGAAGAATATGCAAAAATTTTCCTGCAAACAGCAAAGTTCGAGGTG TTATTGTCGGCATGTGCTACAATGCTGAATGGAGGTTCTCCAGCGCACCAGTATGCCCACCCTATGG TTGCCCACCGATACAAGACAATGACAAATTCCGGCACGAATACTATGAATACGCTGGGAGTCGAGA TGTCGTCGGACAAGCAGTAACTAAGGACAGTTCTGGAAATTACCCTCCACAGACGCATGCAAGAAG GCATTGCCAAGCACGGAGCGTAAAGGTTGATCAAGGGGAACTTGTCGCCATTTGCCACGAAAGAA GTGATACTGGTGAAAGTCGGTGGGTGTACTATCATAATATAAAACAGTGCCCAGTTCCTGGCTGTAC CATAACATATAAAGCCAACAGTACCGTTACGTATAAGTATCTCCAATACACAATGTCTCCTGACCAA ACTGTCGTCGCAAAGGAAGCGACACCGCAGAGCGGAGAATACCCTGACGGCACGTTTGCGATAGT CATCTGTAAGATACCGACTAGAATAGGCGAAAAAACAGGGACAATTATTGCACAATGCAGCAATCG CCAATGGAAGCCAGAAAAGTACCAAATCATACCGGAATGCCCTGGGCGTGAATGCGCCCCTCTTAC GGACAACGACACCGTAAGATATGAGTATCTCAATATATCACGCACAGGCTACTATACACTAAGCAC CTCCACTGTAAAGCCACGGTCTGGAAAATACCCCGAACGCACGTCTGCAAAGATGTTTTGTAAGAA GGCAACTGGCGATAACAAAAACTTAGGAGAAATCGCTGGAAGATGCAGCAAAGGCGAATGGGTGA AGGAGAATGAAGCGACGGTGCTACAATGTCCTATTCTTGGATGCTTACCGTTGGCCGAGAACGACA CCGTGGAATACAAGTACTTTAAAAGTGAGCAAACAAGTAACGATTTCGCAAATATTGCCGTACTAGA CTGGAGTGGGAAATTCGCTGTGGGCTCGTATGCTCGGAGGATTTGCAAGAAACTAGACGTAAACAG CAGGGCTCAAGGGGATATCTCCAAATGCGGCGCGAACGGATGGGTGCACCGCAACACATGGCCAT GTCCACCACAGGGGAGCTGCAATATAAACGAGGTTTATTTGGATCTGAAATTCACCTCAACTATAGT CGATACACGTCTTGCGGTGGCAGTATACAAGAAACCTGATACTGTTTCTACATTTTACGGACCTGGTT CGAAAATACAAGCACTTTGCAAAGGTAACCCGGCAGATCTTGAATGCTTTGAAGGTGGATGGCAAG GACGTCGTCATGCCGAGAATGAGAGAAAATTCGCTAAAATACGATGCACTGATAGCGGAGTCACCT ATGACGAATAA Amino Acid MLLIVVIGLLEASAAGDNSCMPLSEETDTYQYFAQTSNKEETPARKDSSGMYPEYTHVKRFCKGLHGEDK Sequence TGEFIGLCHRSEWVYYMGVQECRDRRCSPLSESDTVSYEYRKATLNSSRISYDTSANPDDSGKYPELTYIR RICKNFPANSKVRGVIVGMCYNAEWRFSSAPVCPPYGCPPIQDNDKFRHEYYEYAGSRDVVGQAVTKD SSGNYPPQTHARRHCQARSVKVDQGELVAICHERSDTGESRWVYYHNIKQCPVPGCTITYKANSTVTYK YLQYTMSPDQTVVAKEATPQSGEYPDGTFAIVICKIPTRIGEKTGTIIAQCSNRQWKPEKYQIIPECPGREC APLTDNDTVRYEYLNISRTGYYTLSTSTVKPRSGKYPERTSAKMFCKKATGDNKNLGEIAGRCSKGEWVK ENEATVLQCPILGCLPLAENDTVEYKYFKSEQTSNDFANIAVLDWSGKFAVGSYARRICKKLDVNSRAQG DISKCGANGWVHRNTWPCPPQGSCNINEVYLDLKFTSTIVDTRLAVAVYKKPDTVSTFYGPGSKIQALCK GNPADLECFEGGWQGRRHAENERKFAKIRCTDSGVTYDE Comments Larval, not adult, parasite product. Matches TGM 1-196 (72 changes, 63.3% identity incl. 9/9 Cys) and 197-422 (only 63 identities, 27.9% identical, incl 11/13 Cys) with 175-aa insertion (incl. 6 cysteines) TGM-g Assembly Hp_I03145_IG00345_L1870 Nucleotide TTTTGTCCAAAGAATCGTTTATTCAACTGTGGCAAACACTGAGTTTATTCGTCATAGGTGACTCCGCT Sequence ATCAGTGCATCGTATTTTAGCGAATTTTCTCTCATTCTCGGCATGACGACGTCCTTGCCATCCACCTTC AAAGCATTCAAGATCTGCCGGGTTACCTTTGCAAAGTGCTTGTATTTTCGAACCAGGTCCGTAATAT GGTGAAACAGTACCAGGTTTCTGGTATACTGCCACCGCAAGACTTGTATGGACTATAGTTGAGGTGA ATTTCTGATCCAGAAAAACATCGCTTATATCGCAACTCCCGCGTGGGGGGCATGGCCATGTGTTGCT GTGCTCCCATCCGTTCTCCCCGCATTTGGAGATATCCCCTTGAGCCTCGCTTTTTTCGTCCAGTTCCTT GCAAATCCTCCAAGCATACGAGCCCACAGCGAACTTTCCACTCCAATCTAGTGGGGCAATATTTTCG TGCGCGATATTTGGATGTTCACTTTTGAAGTACTTGTATTCCACGGTGTCGTTCTCGTGCAACGGTAG GCATCCAAGAATAGGACATTGTAGCACCGTCGCTTCATTCTCCTTCACCCATTCGCCTTTGCTGCATC TTCCAGCGATTTCCCCTAAGTTTTTGTTATCGTCAGTTGCCTTCTTACAAAACATCTTTGCAGACGTGC CTTCGGGGTATTTTCCAGACCGTGGCTTTACCTTGGAGGTGCTTAGTGTATAGTAGCCTGTGCGTGAT ATATTGAGATACTCATATCTTACGGTGTCGTTGTCCGTAAGAGGGGCGCATTCACGCCCAGGGCATT CCGGTACGATTTGGTACTTTTCCGGCTTCCATTTGCGATTGCTGCATTGTGCAAAAATTGTCCCTGTTT TTTCGCCTATTCTAGTCGGTATCTTACAGATGACTATCGCAAACGTGCCGTCAGGGTATTCTCCGCCC TGCGGTGTCGCTTCCTTTGCGACGACAGTTTGGTCAGGAGATATTGTGTATTGGAGATACTTATACGT AACGGTACTGTTGTCTTCATATGCTGTGGTACAGCCAGGATCTGGGCATTGTTTTATATTAAGATAGT ACACCCACCGACTTTCACCAGTACTATCTCTTTCGAGGCAAATGGCGACAAATTCCCCTTGACCATC CTTTCCGCTCCCTGCGCTGCAACGCCTTCTTGCATGCGTCTGTGGAGGGTAATTTCCAGAACTGTCCT TAGTTGCTGCTGGTCCGAGTTTTTCTCGACTCCCAGCGTATTTATAGTATTCGTGCCGGAAATTGTCA TTGTCTTGTATCGGTGGGCAACCGTAGGGTGGGCATTCTGGTGTGCTGGAAAACCTCCACTCAGCAT TGTAGCACATGCCGACAATGAGACCTCGAATTTTGCTGTCTACAGAAAAATTTTTGCAGGTTCTTCTA ATGTATGTTAGTTCAGGATATTTTCCGCTGCTATCTGGATTGGCATTTGTATCATAACTGATTCTCGAA GAATTTAGAGTAGCTTTACGATACTCGTAGCTGACAGTATCGCTCTCAGACAACGGCGAGCATCTAC GGTCTCGGCATTCTTTTACTCCCATATAATAGACCCATTCTGATCGATGGCACAGGCCGATGAACTC ACCTGTCTTATCTTCTCCATGCAATCCTTTACAAAACCGTTTCACGTGCGTATACTCAGGATACATTCC AGAACTGTCCTTTCGCGCAGGTGTTTCCTCTTTATTAGAAGTTTGTGCAAAATATTGGTAGGTGTCAG TTTCTTCAGATAATGGCATGCAGCTGTTGTCGCCTGCAGCCGAAGCCTCGAGAAGGCCAATTACTAC AATCAGCAGCATTTCACTCAAACTTGGGTAATTAAACC Nucleotide ATGCTGCTGATTGTAGTAATTGGCCTTCTCGAGGCTTCGGCTGCAGGCGACAACAGCTGCATGCCAT Sequence TATCTGAAGAAACTGACACCTACCAATATTTTGCACAAACTTCTAATAAAGAGGAAACACCTGCGCG ORF AAAGGACAGTTCTGGAATGTATCCTGAGTATACGCACGTGAAACGGTTTTGTAAAGGATTGCATGG AGAAGATAAGACAGGTGAGTTCATCGGCCTGTGCCATCGATCAGAATGGGTCTATTATATGGGAGT AAAAGAATGCCGAGACCGTAGATGCTCGCCGTTGTCTGAGAGCGATACTGTCAGCTACGAGTATCG TAAAGCTACTCTAAATTCTTCGAGAATCAGTTATGATACAAATGCCAATCCAGATAGCAGCGGAAAA TATCCTGAACTAACATACATTAGAAGAACCTGCAAAAATTTTTCTGTAGACAGCAAAATTCGAGGTC TCATTGTCGGCATGTGCTACAATGCTGAGTGGAGGTTTTCCAGCACACCAGAATGCCCACCCTACGG TTGCCCACCGATACAAGACAATGACAATTTCCGGCACGAATACTATAAATACGCTGGGAGTCGAGA AAAACTCGGACCAGCAGCAACTAAGGACAGTTCTGGAAATTACCCTCCACAGACGCATGCAAGAA GGCGTTGCAGCGCAGGGAGCGGAAAGGATGGTCAAGGGGAATTTGTCGCCATTTGCCTCGAAAGA GATAGTACTGGTGAAAGTCGGTGGGTGTACTATCTTAATATAAAACAATGCCCAGATCCTGGCTGTA CCACAGCATATGAAGACAACAGTACCGTTACGTATAAGTATCTCCAATACACAATATCTCCTGACCA AACTGTCGTCGCAAAGGAAGCGACACCGCAGGGCGGAGAATACCCTGACGGCACGTTTGCGATAG TCATCTGTAAGATACCGACTAGAATAGGCGAAAAAACAGGGACAATTTTTGCACAATGCAGCAATC GCAAATGGAAGCCGGAAAAGTACCAAATCGTACCGGAATGCCCTGGGCGTGAATGCGCCCCTCTTA CGGACAACGACACCGTAAGATATGAGTATCTCAATATATCACGCACAGGCTACTATACACTAAGCA CCTCCAAGGTAAAGCCACGGTCTGGAAAATACCCCGAAGGCACGTCTGCAAAGATGTTTTGTAAGA AGGCAACTGACGATAACAAAAACTTAGGGGAAATCGCTGGAAGATGCAGCAAAGGCGAATGGGTG AAGGAGAATGAAGCGACGGTGCTACAATGTCCTATTCTTGGATGCCTACCGTTGCACGAGAACGAC ACCGTGGAATACAAGTACTTCAAAAGTGAACATCCAAATATCGCGCACGAAAATATTGCCCCACTA GATTGGAGTGGAAAGTTCGCTGTGGGCTCGTATGCTTGGAGGATTTGCAAGGAACTGGACGAAAAA AGCGAGGCTCAAGGGGATATCTCCAAATGCGGGGAGAACGGATGGGAGCACAGCAACACATGGC CATGCCCCCCACGCGGGAGTTGCGATATAAGCGATGTTTTTCTGGATCAGAAATTCACCTCAACTAT AGTCCATACAAGTCTTGCGGTGGCAGTATACCAGAAACCTGGTACTGTTTCACCATATTACGGACCT GGTTCGAAAATACAAGCACTTTGCAAAGGTAACCCGGCAGATCTTGAATGCTTTGAAGGTGGATGG CAAGGACGTCGTCATGCCGAGAATGAGAGAAAATTCGCTAAAATACGATGCACTGATAGCGGAGTC ACCTATGACGAATAA Amino Acid MLLIVVIGLLEASAAGDNSCMPLSEETDTYQYFAQTSNKEETPARKDSSGMYPEYTHVKRFCKGLHGEDK Sequence TGEFIGLCHRSEWVYYMGVKECRDRRCSPLSESDTVSYEYRKATLNSSRISYDTNANPDSSGKYPELTYIRR TCKNFSVDSKIRGLIVGMCYNAEWRFSSTPECPPYGCPPIQDNDNFRHEYYKYAGSREKLGPAATKDSSG NYPPQTHARRRCSAGSGKDGQGEFVAICLERDSTGESRWVYYLNIKQCPDPGCTTAYEDNSTVTYKYLQ YTISPDQTVVAKEATPQGGEYPDGTFAIVICKIPTRIGEKTGTIFAQCSNRKWKPEKYQIVPECPGRECAPLT DNDTVRYEYLNISRTGYYTLSTSKVKPRSGKYPEGTSAKMFCKKATDDNKNLGEIAGRCSKGEWVKENEA TVLQCPILGCLPLHENDTVEYKYFKSEHPNIAHENIAPLDWSGKFAVGSYAWRICKELDEKSEAQGDISKC GENGWEHSNTWPCPPRGSCDISDVFLDQKFTSTIVHTSLAVAVYQKPGTVSPYYGPGSKIQALCKGNPA DLECFEGGWQGRRHAENERKFAKIRCTDSGVTYDE Comments Larval, not adult, parasite product. TGM-h Assembly Hp_I18123_IG10067_L851 Nucleotide TTTATATTCAAAAATATTTATGCACTTCAGCTGTGAAGACTGTCTCATTCGCAAAGCGCTTTTCTGCCG Sequence TCCGCTGTAACCCAATGGCCGCCGTCAGCACATGTTAACTTCACAGATTCACCTTTGCACTTCGCAG TGACTCTCGAACCGGGACGATAATGCCTGTATGTATTGTCTTCGGAAAATTTTGCTGGCTGATTTACC CAGTATGAAATATCACTGAACATTAGCTTCTCAAGCAGATATTCTTCGTCACATCCTGCTGCGAATCG ACAGTCTGGCAAATTTTTAAGCTTCCACTCTGAATCCAAGCACTCTGCGTAGGTCTCCGCTTGAGCTA GACTATTCCTTGGAAGCTCATTGCAATACCTTCTAACTAGTGTTTGTTCGGGATACTTTCCGCTTCCGT CCGGGTTTGTAAGTGTACCCATGCCCTTTCCTTCAGCCGCTTTTGTGTAATATTCATAGCTCACAGAA ACACTCTTTTTCAGAGGCTCGCACCGAGGGTCCGGGCATGCCGTTATATTACGATAGTACATCCATT GACTTCCACGTCTTTGGTAGCAAATGGCGACAAATACCCCTGGGTCAGCTTTTTTACTGAGAGCCTT ACAATGCGCTCTTGCATGCGTCTGCGGAGGATAATTTCCAGAAGTGTCCTTAGTTGCTGCTTCCCCG ACGGTGTGACGATTTTCAACGTATTCGTAATATTCGTACCACACAGTTTCATTGTCCGGAAGAGGTA GGCAACTTGATCCAACAGCTTCAACCAGTCCGATAGCAACGAATAATAGGAGCATCTTGATGGAGC GTCGATGAGCACAATGGTCTCAAACTTGGGTAATTAAACC Nucleotide ATGCTCCTATTATTCGTTGCTATCGGACTGGTTGAAGCTGTTGGATCAAGTTGCCTACCTCTTCCGGA Sequence CAATGAAACTGTGTGGTACGAATATTACGAATACGTTGAAAATCGTCACACCGTCGGGGAAGCAGC ORF AACTAAGGACACTTCTGGAAATTATCCTCCGCAGACGCATGCAAGAGCGCATTGTAAGGCTCTCAG TAAAAAAGCTGACCCAGGGGTATTTGTCGCCATTTGCTACCAAAGACGTGGAAGTCAATGGATGTA CTATCGTAATATAACGGCATGCCCGGACCCTCGGTGCGAGCCTCTGAAAAAGAGTGTTTCTGTGAG CTATGAATATTACACAAAAGCGGCTGAAGGAAAGGGCATGGGTACACTTACAAACCCGGACGGAA GCGGAAAGTATCCCGAACAAACACTAGTTAGAAGGTATTGCAATGAGCTTCCAAGGAATAGTCTAG CTCAAGCGGAGACCTACGCAGAGTGCTTGGATTCAGAGTGGAAGCTTAAAAATTTGCCAGACTGTC GATTCGCAGCAGGATGTGACGAAGAATATCTGCTTGAGAAGCTAATGTTCAGTGATATTTCATACTG GGTAAATCAGCCAGCAAAATTTTCCGAAGACAATACATACAGGCATTATCGTCCCGGTTCGAGAGT CACTGCGAAGTGCAAAGGTGAATCTGTGAAGTTAACATGTGCTGACGGCGGCCATTGGGTTACAGC GGACGGCAGAAAAGCGCTTTGCGAATGA Amino acid MLLLFVAIGLVEAVGSSCLPLPDNETVWYEYYEYVENRHTVGEAATKDTSGNYPPQTHARAHCKALSKKA Sequence DPGVFVAICYQRRGSQWMYYRNITACPDPRCEPLKKSVSVSYEYYTKAAEGKGMGTLTNPDGSGKYPEQ TLVRRYCNELPRNSLAQAETYAECLDSEWKLKNLPDCRFAAGCDEEYLLEKLMFSDISYWVNQPAKFSED NTYRHYRPGSRVTAKCKGESVKLTCADGGHWVTADGRKALCE Comments Larval, not adult, parasite product. TGM-i Assembly Hp_I18218_IG10162_L844 Nucleotide GTTTAATTACCCAAGTTTGAGAACTAACGCATTTAAGTTAAATGTGGTTCTCCCTAATTGCAGTCGCA Sequence GTTTTCAACGTTGCAGGAGCAAGTGATGGTTGCCTGCCACTATCTGAGGAAACGGCCACTTATGAAT ATTATGCATACAGCGGGAGTCGGTATGTTGACGGTAACCCCACAGAAAAGGACAGTTCCGGAAGGT ATCCTCATGGCACACATGCCAAGCGATTTTGCAAAGGCTCAGATGAAGAGGCAGGGTTGTTCGTAG CCATATGCGTTAAATATAGATGGGTGTACTACAAGGACGTAAAGCCGTGTCCAGACTTTAGGTGTCA ACCGCTGACGCCAAATGAAACTATCAGCAATTACCAGTACCTCAAAGAAACCACCAATTCTGGAGG AGAAAGCTTTGAAGTCGTCCAACCGGATGCCGACGGAAAATATCCTGAGCTGACGTACATAAGGAG AACGTGCAATGAGTTTCCCACAGACAGAAAGCTACAAAGAGATATCGCCGGCCTTTGCTACAAAGC CGAATGGTTTCTACGAACCTGTCCGACTCCCGGAAATTGCTACGACGACGATATACGCACAAAGTT GAAGTACCAAGGATATTCATTTGACTATGAAACTGCCGAAGTGACTTACTCATTCGGTAATGACGGA GCGCATTATTTCATAGAGGGCTCTCAGGTCACAGGAATTTGTAACGGTTATCAAGTACCCCTATGGT GCCAGGATGGTGAATGGATCGGAGAGGTAAAGAACATTTCCTGCGATATGATGAACGCACAGTAG CATCGTACAGCCAGTGTCTGCGGGACACAATAAATATTTTATTTCTT Nucleotide ATGTGGTTCTCCCTAATTGCAGTCGCAGTTTTCAACGTTGCAGGAGCAAGTGATGGTTGCCTGCCAC Sequence TATCTGAGGAAACGGCCACTTATGAATATTATGCATACAGCGGGAGTCGGTATGTTGACGGTAACC ORF CCACAGAAAAGGACAGTTCCGGAAGGTATCCTCATGGCACACATGCCAAGCGATTTTGCAAAGGCT CAGATGAAGAGGCAGGGTTGTTCGTAGCCATATGCGTTAAATATAGATGGGTGTACTACAAGGACG TAAAGCCGTGTCCAGACTTTAGGTGTCAACCGCTGACGCCAAATGAAACTATCAGCAATTACCAGTA CCTCAAAGAAACCACCAATTCTGGAGGAGAAAGCTTTGAAGTCGTCCAACCGGATGCCGACGGAA AATATCCTGAGCTGACGTACATAAGGAGAACGTGCAATGAGTTTCCCACAGACAGAAAGCTACAAA GAGATATCGCCGGCCTTTGCTACAAAGCCGAATGGTTTCTACGAACCTGTCCGACTCCCGGAAATTG CTACGACGACGATATACGCACAAAGTTGAAGTACCAAGGATATTCATTTGACTATGAAACTGCCGA AGTGACTTACTCATTCGGTAATGACGGAGCGCATTATTTCATAGAGGGCTCTCAGGTCACAGGAATT TGTAACGGTTATCAAGTACCCCTATGGTGCCAGGATGGTGAATGGATCGGAGAGGTAAAGAACATT TCCTGCGATATGATGAACGCACAGTAG Amino Acid MWFSLIAVAVFNVAGASDGCLPLSEETATYEYYAYSGSRYVDGNPTEKDSSGRYPHGTHAKRFCKGSDE Sequence EAGLFVAICVKYRWVYYKDVKPCPDFRCQPLTPNETISNYQYLKETTNSGGESFEVVQPDADGKYPELTYI RRTCNEFPTDRKLQRDIAGLCYKAEWFLRTCPTPGNCYDDDIRTKLKYQGYSFDYETAEVTYSFGNDGAH YFIEGSQVTGICNGYQVPLWCQDGEWIGEVKNISCDMMNAQ Comments Larval, not adult, parasite product.
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