NEW SEROLOGICAL MARKER FOR THE LATENT FORM OF TOXOPLASMOSIS
20220390449 · 2022-12-08
Inventors
- Céline DARD (La Tronche, FR)
- Mohamed-Ali HAKIMI (La Tronche, FR)
- Hervé PELLOUX (La Tronche, FR)
- Mariee-Pierre BRENIER-PINCHART (La Tronche, FR)
- Christopher SWALE (La Tronche, FR)
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
In the present invention, inventors report the characterization of BCLA (Brain Cyst Load-associated Antigen), a protein exclusively expressed during the bradyzoite stage of the parasite. In cysts directly purified from the brain of mice, the protein is distributed within and at the surface of the cyst. ELISA antibody capture using a combination of serologically reactive BCLA peptides and a recombinantly expressed c-terminal domain (rBCLA) constitutes an efficient serological marker of latent infection with a high sensitivity that is clearly and exclusively correlated with the presence of cysts in the brain of mice Antibodies directed against BCLA antigen have been detected in human patients with enriched titers in patients qualified as seropositive to Sag1 or tachyzoite related antigens. Further correlation in humans between anti-BCLA IgG synthesis and cysts is brought by significantly stronger recorded titers in pathological panels strongly related to the presence of cyst. Furthermore, newborn infants with a confirmed congenital toxoplasmosis display significantly higher anti-BCLA IgGs at birth when compared to their mother, suggesting a specific in-utero neosynthesis of such IgGs. Thus the invention relates to a new Toxoplasma gondii protein, hereafter referred as BCLA, a new serological marker whose expression is restricted to the latent form of Toxoplasmosis (bradyzoite/cyst). This specific protein and its antigenic fragments can be used to detect autoantibodies in the sera of patient for the diagnosis of the latent form of Toxoplasmosis. The invention also relates to derived antibodies, generated by BCLA immunisation that specifically binds this new protein.
Claims
1. A polypeptide or an isolated polypeptide comprising: the Toxoplasma gondii polypeptide BCLA amino acids sequence (SEQ ID NO: 1); (ii) the BCLA C-terminal antigenic domain amino acids sequence (SEQ ID N 2); (iii) a BCLA internal repeated domain amino acids sequence selected from the group consisting of: TABLE-US-00020 (SEQ ID NO: 4) TgR1, (SEQ ID NO: 5) TgR2, (SEQ ID NO: 6) TgR3, (SEQ ID NO: 7) TgR4, (SEQ ID NO: 8) TgR5, (SEQ ID NO: 9) TgR6, (SEQ ID NO: 10) TgR7, (SEQ ID NO: 11) TgR8, (SEQ ID NO: 12) TgR9, (SEQ ID NO: 13) tgR10, (SEQ ID NO: 14) TgR11, (SEQ ID NO: 15) TgR12 and (SEQ ID NO: 16) TgR13; (iv) an amino acid sequence substantially homologous to the sequence of (i), to (ii) or (iii); or (v) a fragment of at least 9 consecutive amino acids of the sequence of (i), (ii), (iii) or (iv).
2. The isolated polypeptide according to claim 1, which comprises a fusion between two peptides fragments of any an amino acid sequence of (i), to (ii), (iii), (iv) or (v).
3. The isolated polypeptide according to claim 1 which is selected from the group consisting of: TABLE-US-00021 (i) (SEQ ID NO: 55) MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP, (ii) (SEQ ID NO: 56) AAGSMEKDKLVLPGE. an amino acid sequence substantially homologous to the sequence of (i) or (ii), or (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i), (ii) or (iii).
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. A method for detecting and/or evaluating an amount of the polypeptide according to claim 1, in a biological sample, comprising contacting the biological sample with an antibody that specifically binds to the polypeptide.
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. A method for diagnosing or confirming a diagnosis of and treating a latent form of Toxoplasmosis in a patient who is suffering, or is suspected to be suffering, from the latent form of Toxoplasmosis, comprising: a) obtaining a biological sample from the patient, b) detecting, in the biological sample, antibodies toward the T. gondii polypeptide according to claim 1, and c) treating the patient with a folic acid antagonist and/or an antibiotic if the antibodies are detected.
15. An in vitro method for diagnosing or confirming a diagnosis of and treating congenital Toxoplasmosis in a patient who is suffering, or is suspected to be suffering, from a congenital Toxoplasmosis, comprising: a) obtaining a biological sample from the patient, b) detecting, in the biological sample, antibodies toward a T. gondii polypeptide according to claim 1; and c) treating the patient with at least one folic acid antagonist and/or at least one antibiotic, if the antibodies are detected.
16. A method for detecting bradyzoite cyst, and/or evaluating its amount in a subject, wherein said method comprises a) detecting in a fluid sample of the subject immunoreactivity toward a T. gondii polypeptide according to claim 1; wherein immunoreactivity toward the T. gondii polypeptide is indicative of the presence and/or amount of the bradyzoite cyst in said subject.
17. The method according to claims 13 to 16 wherein said biological sample is a fluid sample.
18. A method for treating a patient infected with latent form of Toxoplasmosis who shows immunoreactivity toward a T. gondii polypeptide of claim 1, comprising administering to the patient at least one folic acid antagonist and/or at least one antibiotic, or a pharmaceutical composition comprising said compounds the at least one folic acid antagonist and the at least one antibiotic compound.
19. The polypeptide or the isolated polypeptide of claim 1, wherein the amino acid sequence is at least 80% identical to the sequence of (i), (ii) or (iii).
20. The method of claim 14, wherein the at least one folic acid antagonist is pyrimethamine and the at least one antibiotic is sulfadiazine or spiramycin.
21. The method of claim 15, wherein the at least one folic acid antagonist is pyrimethamine and the at least one antibiotic is sulfadiazine or spiramycin.
Description
FIGURES
[0338]
[0339] (a) Quantitative proteome-wide analysis by LC-MS/MS after TgHDAC3 inhibition with FR235222 reveals the expression of bradyzoite-specific proteins among them BCLA. The volcano plot shows the distribution of T. gondii proteins comparing untreated (DMSO, 0.1%) versus FR235222 treated (90 nM) primary human fibroblasts infected with type II (PruΔku80) strain. The log 2 ratios (x-axis) for proteins counts were obtained by dividing intensities of FR235222 treated samples by intensities of DMSO-treated samples (control). The down- and up-regulated proteins are shown in red spots on respectively the left and the right side of the graph. The vertical black lines indicate log 2 fold changes values. The horizontal dashed black lines distinguish the proteins (red spots) showing at least a 2-fold abundance change with p-values <0.01. (b) Bar graphs showing the expression (fragments per kilobase of transcript per million mapped reads [FPKM] values) of BCLA gene during: acute (tachyzoite) or chronic (cyst-enclosed bradyzoites) infection in mice, in various cat entero-epithelial stages (EES) samples (EES1: very early EES; EES2: early EES; EES3: mixed EES; EES4; late EES; EES5: very late EES) from day 3 to day 7 after oral infection with T. gondii cysts (CZ clone H3), and in cysts from mouse brain and in in vitro cultivated tachyzoites. BCLA is only expressed during the chronic stage in cyst-enclosed bradyzoites and is not found in cat entero-epithelial stages (EES) from EES1 to EES5 (data source: www.ToxoDB.org). (c) A genome browser (IGB) screenshot of BCLA locus (in magenta) on chromosome Ib of T. gondii, showing reads for two histone marks (H3K14ac, H3K9me3), TgHDAC3, TgCRC230 as well as RNA-seq (expressed in FPKM, in black). The y-axis depicts read density. This view shows an enrichment of H3K14ac, H3K9me3, TgHDAC3 and TgCRC230 at BCLA gene. (d) Left panel: CRISPR-mediated gene disruption of TgHDAC3 leads to TgHDAC3 signal suppression when monitored by immunofluorescence assay. Right panel: CRISPR-mediated gene disruption of TgHDAC3 triggers BCLA overexpression when monitored by immunofluorescence assay.
[0340]
[0341] (a) Chart illustration showing the disorder score as a function of protein amino acid position (generated via the IUPred server). ANCHOR2 and IUPred2 algorithm results are displayed in blue and red respectively. The C-terminal domain end of BCLA (residues 1089 to 1275, hereafter referred as rBCLA) is predicted as structured, as opposed to the rest of the protein containing core repeated motifs. (b) BCLA protein encoded by a type II (ME49) T. gondii strain displays 13 repetitions in its structure (TgR1 to TgR13). In house antibodies directed against two peptides (peptides 1 and 2) contained in these repetitions were made by Eurogentec company. (c) BCLA expression monitoring by western-blot using the home-made antibodies raised against the two BCLA-derived peptides shows up-regulation of BCLA after FR235222 treatment compared to DMSO (control).
[0342]
[0343] (a) Quantification of the intensity of BCLA in each PV following FR235222 stimulation. Each symbol marks the BCLA density of a single PV. The results are represented as mean±standard deviations from two independent experiments; the number of PVs quantified was at least 70. Asterisks indicate statistical significance when comparing each individual FR235222-treated strain and the corresponding control (DMSO, 0.1%) as determined by an unpaired two-tailed Student's t-test (Mann Whitney test) (**** p<0.0001; NS, not significant).
[0344]
[0345] (a) Evaluation of the percentage of invasion (left panel) in HFFs as well as the intracellular proliferation rate (right panel) of 76k-GFP-luc-Δbcla tachyzoites cultivated in vitro compared to the WT strain. The % of HFFs invasion is quite similar in both strains but the deletion of BCLA induces a 30% decrease of intracellular proliferation. The results are represented as mean±standard deviations from two independent experiments. Asterisks indicate statistical significance when comparing 76k-GFP-luc-Δbcla and 76k-GFP-luc by the Mann Whitney test (an unpaired two-tailed Student's t-test), ** p<0.01; NS, not significant.
[0346]
[0347] (a) Comparison of the virulence of 76k-GFP-luc-Δbcla strain with its parental strain 76k-GFP-luc (WT) in Balb/c and NMRI mice. Balb/c mice (n=20) and NMRI mice (n=43) were inoculated by intraperitoneal (i.p.) injection with 10.sup.4 and 10.sup.6 tachyzoites respectively, and survival was monitored during 35 days. Significance was tested using Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. Mice infected with Δbcla tachyzoites survived to infection with the same time frame than the WT strain (NS, not significant). (b) Evaluation of the ability of Δbcla strain compared to the WT strain to migrate through the brain blood barrier and to form T. gondii cysts in brains of mice chronically infected with T. gondii. Brains of NMRI mice and Balb/c mice that survive to challenge presented in (a) were harvested and tested by quantitative PCR±cyst count using microscopy to evaluate the parasitic load and the number of cysts respectively. The results are represented as mean±standard deviations from at least two independent experiments. Statistical significance was tested by an unpaired two-tailed Student's t-test (Mann Whitney test). Mice infected with Δbcla strains show a trends in decrease (but not significant, NS) of the parasitic load and the number of cysts in brain.
[0348]
[0349] The cyst morphology of Δbcla bradyzoite-containing cysts was compared with those from the parental 76k-GFP-luc (WT) strain. Brains of NMRI mice that survive to challenge presented in
[0350]
[0351] Evaluation of the virulence and infectivity of the 76k-GFP-luc-Δbcla-containing cysts comparing to the 76k-GFP-luc parental strain (WT). C56BL/6 mice (n=6) and NMRI mice (n=20) were orally infected with 46 cysts and 20 cysts respectively of Δbcla or WT strains. Acute response in ilea was observed in C56BL/6 mice 8 days post-infection. Chronic response in brains was evaluated in NMRI mice 8 to 10 weeks post-infection. (a) Parasitic load in ilea quantified by qPCR of C56BL/6 mice orally infected 8 days earlier. Statistical significance between Δbcla and WT strains was tested by an unpaired two-tailed Student's t-test (Mann Whitney test). No significant difference was observed (NS, not significant). (b) qRT-PCR analysis of cytokines (IFNγ, IL-22, IL-18 and IL-1β) and chemokines (CCL2) in ilea of C56BL/6 mice orally infected 8 days earlier. RNA levels were normalized using TBP levels. Mean values±standard deviations are shown. Statistical significance between Δbcla and WT was tested by the Mann Whitney test. No significant difference was observed (NS, not significant). (c) Brains of NMRI mice orally infected 8-10 weeks earlier were harvested and tested by quantitative PCR and cyst count using microscopy to evaluate the parasitic load and the number of cysts respectively. The results are represented as mean±standard deviations from two independent experiments. Statistical significance between Δbcla and WT was tested by the Mann Whitney test. No significant difference was observed (NS, not significant). Mice infected with Δbcla strains show a trends in decrease (but not significant, NS) of the parasitic load and the number of cysts in brain. (d) qRT-PCR analysis of cytokines (TNF-α, IFNγ, IL-6, IL-22) in brains of NMRI mice orally infected 8-10 weeks earlier. RNA levels were normalized using TBP levels. Mean values±standard deviations are shown. Statistical significance between Δbcla and WT was tested by the Mann Whitney test. No significant difference was observed (NS, not significant).
[0352]
[0353]
[0354] Single western-blot strips were loaded with 0.5 μg of rBCLA and tested on sera collected from mice in sub-chronic (21-41 days) or chronic phase (>42 days) of toxoplasmosis. rBCLA only reacts with anti-T. gondii IgG antibodies of mice with sub-chronic or chronic toxoplasmosis following infection by type II cystogenic strains (PruA7, ME49 or 76k-GFP-luc). (a) Immunoblots on sera from Balb/c mice infected by i.p. with 10.sup.3 to 10.sup.6 tachyzoites/mouse of PruA7 (type II) strain during 42 days. The sera are reacting quite proportionally with rBCLA according to the tachyzoite load. (b) Immunoblots on sera from CBA mice infected by i.p. with 10.sup.6 tachyzoites/mouse of ME49 (type II) strain during 80 days. The sera are strongly reacting with rBCLA. (c) Immunoblots on sera from NMRI mice orally infected with 20 cysts of 76k-GFP-luc (type II) strain during 22 months. The sera are strongly reacting with rBCLA. (d) Immunoblots on sera from Balb/c mice infected by i.p. with 10.sup.6 tachyzoites/mouse of 76k-GFP-luc or 76k-GFP-luc-Δbcla (type II) strains during 21 days. The sera from mice infected with 76k-GFP-luc are strongly reacting with rBCLA, whereas those infected with 76k-GFP-luc-Δbcla are barely reacting with rBCLA. (e) Immunoblots on sera from CBA mice infected by i.p. with 10.sup.3 tachyzoites/mouse of RH (type I) strain followed by pyrimethamine (PYR) or sulfadiazine (Sulfa) treatment for 22 days. The sera are very slightly reacting with rBCLA. (f, g) Immunoblot on sera from NMRI mice infected by i.p. with (f) CTG (type III) strain or (g) PruΔku80 (type II) strain by i.p. with 10.sup.6 tachyzoites/mouse for 42 days. The sera are not reacting with rBCLA. (h) Immunoblots on sera from Balb/c mice infected by i.p. with 10.sup.5 tachyzoites/mouse of 76k-GFP-luc (type II) strain and reactivating or not using corticosteroids treatment during 42 days. All the sera are strongly reacting with rBCLA.
[0355]
[0356] (a) Analysis of the proteolysis reaction by SDS PAGE. Coomassie coloured SDS PAGE showing the input sample and all these time points (10, 20 and 50 minutes) for every protease (trypsin, chymotrypsin, elastase and papain). (b) Blotted gel incubated with a positive mouse serum and revealed by anti-mouse IgG antibodies. (c) Blotted gel incubated with anti 6his IgGs coupled to peroxidase. The black arrow shows non degraded rBCLA. Red and blue cursor arrows show recurring N-terminal degradations, showing that rBCLA is quickly degraded by chymotrypsin and partially degraded by elastase, trypsin and papain generating stable fragments around the 17-kDa marker.
[0357]
[0358] Single western-blot strips were loaded with 0.5 μg of rBCLA and tested on mice sera infected with strains isolated from humans or directly on human sera. (a) Immunoblots on sera from Swiss mice i.p. infected with amniotic fluid or placenta from women with suspected (clinically suspected but T. gondii PCR negative on amniotic fluid or placenta) or confirmed (T. gondii PCR positive on amniotic fluid) congenital toxoplasmosis. The sera from mice infected with positive amniotic fluid are strongly reacting with rBCLA. (b) Immunoblots on sera (S) or aqueous humor (HA) from human patients with or without toxoplasmosis infection. Human sera and aqueous humor were randomly selected from the biobank of the Parasitology-Mycology Clinical Laboratory of Grenoble Alpes University Hospital. For each sample, Toxoplasma serological assay was performed using the Vidas® (bioMérieux) and the Architect® (Abbott)) systems, both based on ELISA-derived techniques, and the clinical status was evaluated using the medical records of each patient. Of note, Vidas® is based on rSAG1 antigen, and Architect® on rSAG1 and rGRA8 antigens. The serological results obtained with rBCLA were compared to the serological and clinical status of each patient to evaluate if they were correlated to a specific T. gondii serological and/or clinical status. Sera of patients with (α) proven or suspected ocular toxoplasmosis, (β) toxoplasmosis reactivation during hematological disease (immunosuppression) and (γ) recent primary infection (between 1 and 2 months) are reacting with rBCLA. (δ) Sera from 3 seropositive patients qualified as “past immunity” and 1 serum from a quite recent infection (2.5 months) are not reacting with rBCLA. (ζ) All the sera tested from the seronegative patients are not reacting with rBCLA, showing a good specificity of this antigen in humans.
[0359]
[0360] (a), Schematic representation of the epitope mapped regions in both the repeat n° 4 and rBCLA region. Peptide coverage is displayed as a line representing the individual 15aa peptides above or below the peptidic sequence, with partial numbering displayed. Regions displaying significant or strong reactivity are highlighted in full or dashed boxes respectively and each individual peptidic fragment are marked with (* or **). (b), Epitope mapping of BCLA positive sera. Below, histograms displaying the relative reactivity of peptides on both the core repeat region and rBCLA region, calculated using 5 different positive blots with a negative background subtraction. Above is an example of a dot blot membrane revelation pattern with numbered peptides, performed on a positive human serum. (c), Peptidic dot blots for 5 positive sera and one negative serum with peptide numbering and regions covered. On the right, ELISA titrations for rBCLA and SAG1 (Architect) are shown for these same sera.
[0361]
[0362]
[0363]
[0364] (A-B) Violin plots of BCLA ELISA titrations (in UI) in sera of 23 mothers and respective newborns collected at the delivery (mothers) or between birth and 5.5 months of age (babies). (C-D) Violin plots of Sag1 titrations (Vidas® and Architect® IgG/IgM). The sera were grouped within clinical status categories of “mother-newborn” couples without congenital toxoplasmosis (A and C) and couples with confirmed congenital toxoplasmosis (B and D). On the top of each panel the values of the mean±SD are represented while the difference between medians was calculated with Mann Whitney test.
EXAMPLE 1
[0365] Materials and Methods
[0366] Host cells and parasites culture. HFF primary cells (Bougdour et al., 2009), RAW264.7, L929, HCT116, A549 and HEK293 cells were cultured in Dulbecco's Modified eagle Medium (DMEM) (Thermo fischer Scientific, France) supplemented with 10% heat-inactivated fetal Bovine Serum (FBS) (Invitrogen), 10 mM (4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid) (HEPES) buffer pH 7.2, 2 mM L-glutamine and 50 μg/ml of penicillin and streptomycin (Thermo Fisher Scientific). Cells were incubated at 37° C. in 5% CO2. The following Toxoplasma strains were used in this study: type I (RH, GT1), type II (ME49), type III (CTG), atypical (COUG), Neospora caninum; RHΔku80 (Huynh and Carruthers, 2009), PruΔku80 (Fox et al., 2011), PruA7 (Saeij et al., 2007), COUGΔmyr1 (Hakimi, unpublished), PruΔku80Δbcla, PruΔku80-HF-BCLA and 76k-GFP-luc-Δbcla obtained in this study. All parasite strains were maintained in vitro by serial passage on monolayers of HFFs. T. gondii transfection. T. gondii RHΔku80, PruΔku80 and 76k-GFP-luc were electroporated with vectors in cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl2) using a BTX ECM 630 machine (Harvard Apparatus). Electroporation was performed in a 2 mm cuvette at 1.100 V, 25Ω and 25 μF. Stable transgenic parasites were selected with 1 μM pyrimethamine, single-cloned in 96 well plates by limiting dilution and verified by immunofluorescence assay.
[0367] Cas9-mediated C-terminal tagging and gene disruption in T. gondii. The plasmid pTOXO_Cas9-CRISPR was described by (Sangare et al., 2016). The gene of interest (GOI) was BCLA (TGME49_209755) for both C-terminal tagging (HA-Flag (HF)) and gene disruption (KO) using the CRISPR/Cas9 system. Four oligonucleotides corresponding to BCLA were cloned using the Golden strategy. Briefly, primers TgBCLA-CRISP_FWD and TgBCLA-CRISP_REV containing the sgRNA targeting TgBCLA genomic sequence were phosphorylated, annealed and ligated into the linearized pTOXO_Cas9-CRISP plasmid with Bsal, leading to pTOXO_Cas-CRISPR::sgTgBCLA. T. gondii tachyzoites were then transfected with the plasmid and grown on HFF cells for 18-36 hours.
[0368] Cloning oligonucleotides used in this study were:
TABLE-US-00015 TgBCLA-KO-CRISP-FWD: (SEQ ID No 28) 5’-AAGTTGATCACTATTCGTGAAGAAGG-3’ TgBCLA-KO-CRISP-REV: (SEQ ID No 29) 5’-AAAACCTTCTTCACGAATAGTGATCA-3’ TgBCLA-HF-CRISP-FWD: (SEQ ID No 30) 5’-AAGTTGGAACGGCGGTACGGCGACCG-3’ TgBCLA-HF-CRISP-REV: (SEQ ID No 31) 5’-AAAACGGTCGCCGTACCGCCGTTCCA-3’
[0369] FR235222 treatment and induction. FR235222 was provided by Astellas Pharma Inc. (Osaka, Japan) and dissolved into DMSO as described by Bougdour et al., 2009 and the final concentration in culture medium was either 25 ng/mL or 50 ng/mL. The media containing FR235222 was added to infected HFF cells 16 hours after infection for 24 h to 7 days.
[0370] Mice and Experimental Infection. 6-weeks-old BALBC/c, CBA, NMRI or Swiss mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). Mouse care and experimental procedures were performed under pathogen free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee of the University Grenoble Alpes (agreement no B3851610006). Female mice were used for all studies. For intraperitoneal (i.p.) infection, tachyzoites were grown in vitro and extracted from host cells by passage through a 27-gauge needle, washed three times in PBS, and quantified with a haemocytometer. Parasites were diluted in Hank's Balanced Salt Solution (Life), and mice were inoculated by the i.p. route with tachyzoites of each strain (in 200 μl) using a 28-gauge needle. For oral gavage of infective cysts, brains from chronically infected mice (76k-GFP-luc and 76k-GFP-luc-Δbcla) were crushed in PBS, the number of cysts was microscopically quantified and the mice were forced fed with 100 μl of brain homogenate containing 20 to 40 cysts using ball-tipped feeding needle. Blood was collected by caudal puncture or by intracardiac puncture when the mice were euthanised. Animal euthanasia was completed in an approved CO2 chamber. For histological analysis of ileum and immunolabeling on histological sections of brains or, the ilea and brains were removed from mice, entirely embedded in a paraffin wax block and cut in 5 μm-thick layers using microtome. For statistical analysis of mice survival data, the Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used.
[0371] Cysts purification. Cysts were isolated from brains of mice chronically infected with the 76k-GFP-luc or the 76k-GFP-luc-Δbcla strains for at least 6 weeks, either using the Percoll gradient method as described previously (Cornelissen et al., 1981), either directly by the cysts using a 10 μl pipet for the dyes experimentation in order not to deteriorate the cyst wall for permeability studies. Neither saponin nor trypsin was added at the end of the experiment.
[0372] Cyst quantification. 5 to 12 weeks post-infection, a brain of each of the recipient mice was homogenized in 2 ml of PBS. Numbers of cysts in three or ten aliquots (20 μl each) of the brain suspensions were counted microscopically. The total number of cysts was determined by enumerating the cysts in a 20-μl aliquot and multiplying by 100. For statistical analysis of cysts quantification differences between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was used.
[0373] Cyst characterization. Images of purified cysts were acquired between slide and slip cover with a fluorescence ZEISS ApoTome.2 microscope. Cysts areas and GFP intensities were measured using the ZEN software (Zeiss). For statistical analysis of cysts areas and GFP-intensity differences between 76k-GFP-luc and 76k-GFP-luc-Δbcla cysts, the non-parametric Wilcoxon-Mann-Whitney test was used.
[0374] Quantitative PCR. The parasite loads in brain or ileum were quantified following DNA extraction (QiAmp DNA mini kit, Qiagen) using the quantitative PCR targeting of the Toxoplasma-specific 529-bp repeat element (Reischl et al., 2003). For statistical analysis of parasitic load differences between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was used.
[0375] qRT-PCR analysis of interleukins in brain and ileum. Total RNA was isolated from brains or ilea using TRIzol (Thermo Fisher Scientific). cDNA was synthesized with random hexamers by using the high Capacity RNA-to-cDNA kit (Applied Biosystems). Samples were analysed by real-time quantitative PCR for appropriate probes (brain: TNF-α, INF-γ, IL-6, IL-22β; ileum: INF-γ, CCL2, IL-22β, IL-18 and IL-1β) using TaqMan Gene Expression Master Mix (Applied Biosystems). RNA levels were normalized using TBP levels. qRT-PCR was repeated for three independent biological replicates of each sample and the mean of the results was used. For statistical analysis of RNA levels between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the non-parametric Wilcoxon-Mann-Whitney test was used.
[0376] Immunofluorescence microscopy. Immunofluorescence assays on in vitro parasites were performed as described previously (Braun et al., 2013). In brief, T. gondii-infected HFF cells grown on coverslips or cysts purified from brains of mice were fixed in 3% formaldehyde for 20 min at room temperature, permeabilised with 0.1% (v/v) Triton X-100 for 15 min and blocked in phosphate-buffered saline (PBS) containing 3% (v/v) bovine serum albumin (BSA). For immunolabeling on histological sections of brains, the brain layers spotted on glass slides were first solvent-dewaxed using toluene for 3*10 min and absolute alcohol for 3*10 min. The slides were then treated with citrate buffer pH 6, heated at 100° C. during 1 hour, rinsed with water for 2*10 min and blocked in PBS containing 3% (v/v) bovine serum albumin (BSA). The cells or brain layers were then incubated for 1 hour with the primary antibodies indicated in the figures followed by the addition of secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes) at a 1:1,000 dilution for 1 hour. Nuclei of both host cells and parasites were stained for 10 min at room temperature with Hoechst 33258 at 2 μg/ml in PBS. Coverslips were mounted on a glass slide with Mowiol mounting medium, images were acquired with a fluorescence ZEISS ApoTome.2 microscope and images were processed by ZEN software (Zeiss).
[0377] Antibodies. Primary antibodies: rabbit anti-BCLA (Eurogentec), mouse anti-HA (Roche, RRID: ab_2314622), rat anti-flag (SIGMA), mouse anti-CC2 (gift from Pr. Louis Weiss), mouse anti-GRA1, mouse anti-GRAS, mouse anti-GRA7. Western blot secondary antibodies were conjugated to alkaline phosphatase (Promega), whereas immunofluorescence secondary antibodies were coupled with Alexa Fluor 488 or Alexa Fluor 494 (Thermo Fisher Scientific).
[0378] Western Blot. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon-P; EMP Millipore) by liquid transfer, and Western blots were probed using appropriate primary antibodies followed by phosphatase-conjugated goat secondary antibodies (Promega). Signals were detected using NBT-BCIP (Amresco).
[0379] DBA lectin labelling on in vitro FR235222 parasites and ex vivo cysts. T. gondii-infected HFF cells grown on coverslips or cysts purified from brains of mice were fixed in 3% formaldehyde for 20 min at room temperature, permeabilised with 0.1% (v/v) Triton X-100 for 15 min and blocked in phosphate-buffered saline (PBS) containing 3% (v/v) bovine serum albumin (BSA). The infected cells or cysts were stained with 1:100-diluted Dolichos lectin for 30 min. The stained vacuoles or cysts were examined with a fluorescence ZEISS ApoTome.2 microscope and images were processed by ZEN software (Zeiss).
[0380] Cyst wall permeability. 76k-GFP-luc and 76k-GFP-luc-Δbcla isolated cysts purified from brains of mice were incubated with different dyes of different size (dextran, Texas Red or Cascade Blue, from 3 000 to 40 000 Da, neutral or anionic lysine fixable) (Promega) in 1:100 dilution. After 20 min of incubation at room temperature, the images were acquired with a fluorescence ZEISS ApoTome.2 microscope and images were processed by ZEN software (Zeiss). A minimum of 5 cysts were analysed for each different dye. Cysts incubated in the absence of the dyes were taken as negative controls.
[0381] Recombinant Expression of the C-Terminal Domain of BCLA (Cter-BCLA)
[0382] Design and cloning. Disorder propensity search (using Dis-EMBL or IUPred) predicts BCLA to be highly disordered throughout most of its sequence including the core repeated motifs. The C-terminal end (approximately from aa 1100 to 1275) is however predicted as structured and may constitute a separate domain. To recombinantly express this domain, the N-terminal boundary was chosen at the methionine 1089 and the original C-terminal end was conserved. DNA synthesis was performed by Genscript to generate a fusion construct composed of Cter-BCLA (1089-1275) with a TEV cleavable N-terminal His-tag (
[0383] Recombinant expression. Transformation was performed using BL21(DE3)-CodonPlus—RIL chemically competent E. coli (Stratagene) which were incubated on ice with 1 μg of the pet30-(a) Cter-BCLA plasmid for 10 minutes, heat shocked at 42° C. for 45 seconds, pre-incubated 45 min in LB at 37° C. then spread on a LB agar plate containing Kanamycin (Kan) and Chloramphenicol (Chlo) and incubated for 12 h. A single colony was then picked to inoculate a LB/Kan/Chlo 50 ml pre-culture grown for 16 h. 5 ml of grown pre-culture were then used to inoculate 1 L flasks of Terrific Broth medium (Formedium) containing Chlo/Kan. Cultures were grown at 37° C. until reaching an OD600 of 0.5-0.8 then induced by adding 0.7 mM IPTG (VWR) and further incubated at 18° C. ON. After incubation, cells were centrifuged 25 min at 3000 G, the supernatant was discarded and the pellet flash frozen in liquid nitrogen and kept at −80° C.
[0384] Lysis. Purification was performed on 3 pellets of 1 L cultures, each resuspended in 50 ml of lysis buffer containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM Beta-mercaptoethanol (BME), 0.2% w/v N-Lauryl Sarkozine and 1 Complete anti protease cocktail (Roche) tab per 50 ml. Lysis was performed using a 10 mini-pulsed sonication (15 sec ON, 30 sec OFF) at 50° amplitude over ice with the lysate never reaching a temperature over 13° C. After sonication, the lysate was centrifuged at 4° C. for 1 h at 15 000 G and the pellet was discarded. All the following steps were subsequent at 4° C. Prior to incubation with 5 mL of pre-equilibrated Ni-NTA resin, the clarified lysate was supplemented with 30 mM Imidazol. Batch incubation was performed for 30 minutes at 4° C. with a gentle stirring. After incubation, the resin was retained on a vertical column then washed with 3*20 ml of wash buffer containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM BME, 0.2% w/v N-Lauryl Sarkosine and 30 mM Imidazol. Direct elution with 1.5 ml fractionations was then performed using a buffer containing 300 mM NaCl, 50 mM Tris pH 8, 5 mM BME and 300 mM Imidazole. Fractions of interest (
[0385] Ion exchange and size exclusion chromatography. The entire sample was then directly pumped through the chromatography system (Akta Pure, GE healthcare) onto a HL-Mono-Q (GE healthcare) 5 ml column pre-equilibrated with the same buffer as for dialysis. The column was washed with 2 column volumes (CV) then eluted by a salt gradient (50 mM to 2M NaCl) over 40 ml, 1.5 ml fractions and 280 nm absorbance monitoring was performed for the entirety of the elution. During the elution, SDS PAGE analysis (
[0386] Ammonium sulphate precipitation. To avoid nucleic acid contamination, an ammonium precipitation was performed by adding 15% w/v of ammonium sulphate (Sigma), gentle rolling at 4° C. for 1 h then 30 minutes of centrifugation at 10 000*G. The supernatant was discarded and the pellet resuspended in the same initial volume of buffer. To clear all ammonium sulphate, the sample was dialysed in the same buffer as for the size exclusion.
[0387] Limited proteolysis to find antigenic sub-fragments within Cter-BCLA. To recover a highly antigenic sub-fragment of Cter-BCLA, a limited proteolysis on the purified sample was undertaken using trypsin, chymotrypsin, elastase and papain (all Sigma Aldrich). Reactions were carried out in 30 μl reaction volume in 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM BME and 0.5 mM MgCl.sub.2. In each reaction, 3 μg of Cter-BCLA were digested by 100 ng of protease (1/30 w/w) over the course of 50 minutes at 37° C. Reactions were stopped at each time point by the addition of 10 μl of SDS PAGE loading buffer followed by 5 minutes heating at 95° C. then kept on ice until loading on the gel.
[0388] Western-blot BCLA serological testing. Single western blot strips were prepared using 15 well 4-12% NuPage gels (Life technologies) loaded with 5 μl of sample at 0.1 mg/ml. The gels were run at 185 v for 40 minutes in MES buffer then electro-transferred at 105 v for 1.5 h on PVDF membranes. The transferred lanes were then cut-out into individualized strips. The strips were then blocked in TTBS with 5% powdered milk (w/v) for 1 h. Serum testing was then performed in TTBS with a dilution of 1/400 of the serum for 1 h at 4° C. The strips were then washed 3 times in TTBS and further incubated 1 h with a 1/7500 dilution of secondary antibody targeting either mouse IgGs or human IgGs and coupled with a phosphatase alkaline enzyme (Promega). Following a 3-time TTBS wash, the blots were revealed by the addition of the chromogenic substrate at RT (Invitrogen). Bands in the positive sera appear within 1 to 5 minutes. In parallel to the serum testing, a single strip was always used as an internal antigen control for each blot set. After blocking, this strip was incubated for 1 h with a peroxidase coupled anti poly-histidine monoclonal antibody (Sigma) diluted 1/2000 in TTBS. After three wash steps in TTBS, the blot was revealed using the SigmaFast DAB with metal enhancers (Sigma). For each series of i.p. or orally infected mice, a serum of at least on mouse of each series was checked for Toxoplasma antibodies using Western blot analysis of the IgG immune response using the commercial kit LD bio Toxoplasma mouse IgG (LD bio), with the same anti-mouse IgG-alkaline phosphatase conjugate and chromogenic substrate previously described for BCLA.
[0389] Human sera. Human sera were retrospectively selected from the biobank collection of the Parasitology-Mycology Clinical Laboratory in Grenoble Alpes University Hospital in France. This biobank is registered with the French Ministry of Health number DC-2008-582. The selected sera were stored for toxoplasmosis serological routine analysis between Jan. 1, 2014 and May 1, 2018. The analyses with Vidas® Toxo IgM and IgG (bioMérieux, France) and Architect Toxo IgG and IgM (Abbott, Germany) were performed in the Parasitology-Mycology Clinical Laboratory of Grenoble Alpes University Hospital
[0390] Protein purification-, immunoblotting- and mass spectrometry-based proteomic analysis. PruΔku80-BCLA-HAFlag infected host HFFs cells extracts containing Flag-tagged protein were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich) for 1 hour at 4° C. Beads were washed with 10 column volumes of BC500 buffer (20% glycerol, 20 mM Tris-HCl pH 8.0, 500 mM KCl, 0.05% NP-40, 100 mM PMSF (phenylmethylsulphonyl fluoride), 0.5 mM DTT and 1× protease inhibitor). Bound peptides were eluted stepwise with 250 g/ml FLAG peptide (Sigma-Aldrich) diluted in BC100 buffer. Protein bands were excised from colloidal blue-stained gels (Thermo Fisher Scientific), treated with DTT and iodoacetamide to alkylate the cysteines before in-gel digestion using modified trypsin (Sequencing grade; Promega). Resulting peptides from individual bands were analysed by online nanoLC-MS/MS (UltiMate 3000 coupled to LTQ-Orbitrap Velos Pro; Thermo Fisher Scientific) using a 25-min gradient. Peptides and proteins were identified and quantified using MaxQuant (version 1.5.3.17) through concomitant searches against ToxoDB (20151112 version), and the frequently observed contaminant database embedded in MaxQuant. Minimum peptide length was set to 7 amino acids. Minimum number of peptides, razor+unique peptides, and unique peptides were all set to 1. Maximum false discovery rates were set to 0.01 at peptide and protein levels.
[0391] Epitope mapping of BCLA repeat and rBCLA. Dot blot peptide assays were custom synthetized by JPT Peptide technologies on cellulose membranes with N-Acetyl moieties on the N-terminus. Two sets of membranes were screened: 1) covering the rBCLA region (res 1089-1275) with a total of 59 peptides, each 15 aa long with an overlap of 12 and an offset of 3; 2) covering repeat 4 (res 446-493) with a total of 18 peptides, each 15 aa long with an overlap of 12 and an offset of 3. Dot blot assays were performed as described by the manufacturer. Briefly, the membranes were first activated 5 min in 100% ethanol then washed 3 times 3 min in DPBS-tween. Blocked O.N at 4° C. in DPBS-Tween 0.5% powdered milk then washed again 3*3 min in DPBS-tween. Tested sera were diluted to 1/400 in DPBS-tween 0.1% BSA and incubated for 3 h at RT with the membrane. Following a 3*3 min DPBS-tween wash, membranes were incubated with anti-IgG peroxidase coupled Ab (Sigma A0170) diluted to 1/100 000 for 2 h at RT. Following a 3*3 min wash in DPBS-tween the membrane was briefly immerged in in the freshly prepared SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) and revealed using the C-Digit (Licor) scanner. Dot intensity was integrated using ImageJ. Dot intensity was integrated using ImageJ. For data analysis of independent dot blots, integrated intensities from every peptide dot [I.sub.(p)] were normalised into enrichment factors Fe.sub.(p) using the baseline integrated intensity of peptide 59[I.sub.(p=59)] which never reacts with any sera. The following can be expressed with the following equation:
[0392] Where p represents the peptide number.
[0393] In order to increase the reactivity score over several independent positive sera blots symbolised as (+), Fe.sub.(p) enrichment scores were summed with each other and to subtract the non-specific reactivity, the same sum was performed on the same number of negative sera peptides, symbolised as (−) and subtracted. Peptide reactivity scores can be expressed though the following equation:
Rs.sub.(p)=[Σ(Fe.sub.(p)).sup.(+)−Σ(Fe.sub.(p)).sup.(−)]
[0394] Where Rs.sub.(p) is the total reactivity score at a specific peptide position.
[0395] BCLA ELISA. Peptide synthesis: The following BCLA peptides were synthetized by Genscript with N-terminal Acetyl groups:
TABLE-US-00016 AB_F: (SEQ ID NNo 55) Nter-MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP- Cter A3_B: (SEQ ID No 56) Nter-AAGSMEKDKLVLPGE-Cter
[0396] Plate preparation: Midisorp plates (Nunc) were coated O.N at 4° C. with rBCLA, peptides AB_F and A3_B all at 2 μg/ml in 100 mM calcium carbonate buffer pH: 9.6 with 100 μl per well. After coating, plates were washed twice with 350 μl of DPBS 0.05% Tween 20 (DPBS/Tween) then blocked for at least 2 h with 300 μL Superblock blocking buffer (ThermoFisher) after which the buffer was removed and the plates dried upside down. Once dried, the plates can be stored for extended periods of time at 4° C. with no loss in serological reactivity.
[0397] Sample preparation: All sera dilutions were prepared in DPBS 0.05% Tween 20, 0.1% BSA no more than 2 h prior to the assay. For both mouse and human tested sera, 1/400 dilutions were prepared. 11 standards were also freshly prepared in both tests, consisting of 10 serial dilutions of a positive frozen stock serum set at 100 UI. Starting at a dilution 1/200 and following a ¾ dilution increment, the following titration points were prepared: 200 UI (1/200), 150 UI (1/266), 112.5 UI (1/356), 84.4 UI (1/474), 63.3 UI (1/632), 47.5 UI, (1/843) 35.6 UI (1/1124), 26.7 UI (1/1498), 20 UI (1/1998), 15 UI (1/2663). A 0 UI standard was prepared with a seronegative serum diluted at 1/400.
[0398] Assay: All the subsequent steps were implemented on the Gemini ELISA automation platform (Stratec) but can also be performed by hand at RT. Dried plates were first washed twice with 350 μl of DPBS/Tween. Dilutions of the tested sera and standards were then distributed in the plates as row duplicates with 100 μl per well. Plates were then incubated 1 h at RT. After the incubation period, plates were washed 4 times with 350 μl of DPBS/Tween, 100 μl of peroxidase coupled secondary antibody dilution (1/50 000 anti-mouse IgG or 1/60 000 anti-human IgG, Sigma Aldrich ref A0168 and A0170 respectively) in DPBS 0.05% Tween 20, 0.1% BSA were then rapidly distributed in all wells. After 1 h at RT, plates were washed 4 times in DPBS tween. Revelation reaction were performed by adding 100 μl of TMB Substrate (Thermofisher ref 34029) for 20 min precisely at RT then stopping the reaction with 50 μl of H2SO4 0.2M followed by 30 sec of mixing. Well absorbance measurement was then performed using the Gemini integrated spectrophotometer at 450 nm.
[0399] Data treatment: Blank subtractions were performed on duplicate blank wells, were no primary antibody/sera was disposed in the well but all subsequent steps (washes, secondary Ab, substrate) were performed. Standard sera dilutions were averaged and fitted with a 4-parameter logistic regression with the upper asymptote value (D.sub.i) fixed at 2.5 AU and all other variables (A.sub.i, B.sub.i, C.sub.i) allowed to fit. From this regression, tested dilution duplicates could have their apparent UI calculated and averaged, if in a duplicate measurement the coefficient of variation was observed above 10%, then the sample would be re-tested. All the ELISA data presented in this work was obtained several times in independent titrations.
[0400] Results
[0401] Quantitative Analysis of the Proteome Response to FR235222 in Tachyzoites Identifies BCLA as a Novel Bradyzoite-Specific Protein
[0402] Specific inhibition of TgHDAC3 by the cyclopeptide FR235222 was shown to disrupt the steady-state level of histone H4 acetylation across the T. gondii genome inducing derepression of stage-specific genes (Bougdour et al., 2009; Sindikubwabo et al., 2017). We have exploited the properties of FR235222 to develop an in vitro cystogenesis system capable of producing the quantities of protein needed for large-scale proteome studies (Farhat D et al., manuscript in preparation). Following a treatment at low dose and for a short period of time of a cystogenic type II (PruΔku80) strain, we performed quantitative proteomics studies and uncovered that the FR235222-treated proteome was significantly enriched in stage-specific proteins including those recognized as restricted to bradyzoite stage (
[0403] BCLA is Secreted into the PV and Associates with the PVM of In Vitro Converted Bradyzoites-Containing Vacuoles
[0404] BCLA is a single open reading frame encoding a 140-kDa protein with a predicted N-terminal signal peptide and a conserved C-terminal region of ˜150 residues that border a central core domain typified by a motif of 48 amino acids repeated 13 times (
[0405] Although BCLA was unequivocally and exclusively identified by mass spectrometry in FR235222-treated samples (
[0406] In fibroblasts hosting tachyzoites expressing a C-terminal HA-Flag-tagged version of bradyzoite-specific markers, BCLA is distinctly detected upon FR235222 stimulation in the vacuolar space and clearly accumulates at the PVM while its expression coincides with the induction of the bradyzoite markers ENO1 and LDH2 (data not shown). Conversely, BCLA was no longer detected in cells infected with tachyzoites genetically engineered to lack BCLA (Δbcla, Table 2), thereby validating the in-house antibodies specificity (data not shown). Finally, when we monitor BCLA dynamics in type I (RHΔku80) and II (PruΔku80) lines expressing the endogenous protein in fusion with the HA-Flag tags, we show that once stimulated by FR235222 HA-tagged BCLA protein is targeted into the vacuolar space and at the membrane regardless of the strain type (data not shown). Thus, the presence of the C-terminal fusion tag does not affect the subcellular localisation of BCLA as it is similar to those seen while using anti-BCLA sera in the untagged strain.
[0407] While exposing different parasite strains of T. gondii to FR235222, we finally uncovered that BCLA signal intensity greatly varies depending of the infecting strains, ranging from a very strong induction in type II (PruΔku80, ME49, 76K-GFP-Luc) strain, rather moderate with type I (GT1 and RHΔku80) and haplogroup 11 (COUG) strains, and surprisingly a faint (if no) signal was detected in cells infected by a type III (CTG) strain (
[0408] BCLA Localizes In Vivo to the Cyst Matrix and Cyst Wall
[0409] The glycosylated cyst wall to which the lectin Dolichos biflorus agglutinin (DBA) binds is the key structural feature that facilitates persistence and oral transmission of T. gondii (Tomita et al., 2013). Here we brought strong evidence of a co-staining of BCLA with DBA exclusively at the membrane surrounding in vitro converted bradyzoites (data not shown) strongly indicating that BCLA, following its delivery into the vacuolar space, accumulates overtime at the wall of immature cysts (based on thin DBA-positive cyst walls).
[0410] Yet, considering that in vitro bradyzoite development in tissue culture does not lead to completely mature cysts, we re-examined the localization of BCLA in bradyzoite-containing cysts isolated from mice chronically infected by T. gondii type II strains. In chronically-infected mice, in-house antibodies raised against BCLA stain the cyst wall as well as the matrix space surrounding bradyzoites (data not shown) The immunofluorescence results did not allow to determine unambiguously, whether the BCLA was located in the inner or in the outer layer of the cyst wall, however and interestingly, non-permeabilized ex-vivo cysts are readily stained by the antibodies, suggesting the outside location of the protein (data not shown) and therefore its exposure to the cytoplasm of the host cell. No signals were detected on cysts hosting Δbcla bradyzoites (data not shown), therefore validating in vivo the specificity of the anti-BCLA antibodies (
[0411] There is not much evidence of extra-vacuolar function of BCLA yet occasionally the protein appears to be exported beyond the vacuolar membrane into the cytoplasm of the host cell (data not shown). Unfortunately, despite many attempts, we did not find the ad hoc conditions underlying BCLA export beyond the PVM to further study in more detail its function in the host cell, if any, as we did for other effectors (Hakimi et al., 2017). Nevertheless, we were able to show that BCLA export was Myr1-independent (data not shown) and as such does not require the T. gondii translocon of exported proteins (Franco et al., 2016). An elegant way to explain the accumulation of the protein in the cytosol of the infected cell is its release after a processing that would take place at the PVM likely under the control of a protease of the host but this remains to be demonstrated. An analysis of the BCLA-associated proteome of an infected and FR235222-stimulated host cell will be performed to determine whether BCLA interactions, if any, with host cell proteins (including protease) occur on the outward-facing side of the PVM or even in the cytoplasm of the infected cell when BCLA is delivered there.
[0412] BCLA is Dispensable for Proper Cyst Function In Vivo
[0413] To determine the function of BCLA in the bradyzoite tissue cyst, we created two parasite line in which the coding region of BCLA was either deleted (PruΔku80Δbcla) or interrupted by a DHFR cassette using Cas9-mediated gene editing (76K-GFP-LucΔbcla) (Table 2). We then investigated pathogenesis and cyst formation. First, BCLA-deficient strains showed no obvious growth phenotype when compared to their parental strains in vitro under tachyzoite conditions (
[0414] BCLA is not Essential for Initiating an In Vivo Infection with Tachyzoites.
[0415] To investigate the importance of BCLA in vivo during acute infection, we compared the parasitic process in BALB/c or NMRI mice infected intraperitoneally (i.p.) of either WT or BCLA-deficient parasites from type II background, the inoculum content ranging from 1×10.sup.4 to 1×10.sup.6 tachyzoites. At 5-8 days after infection, all mice infected with type II BCLA-deficient tachyzoites began to show signs of infection (i.e. weight loss and ruffled fur) and survived to infection with the same time frame that the parental strain 76K, regardless of the inoculum and the genetic background of mice (
[0416] BCLA Deficiency Affects Integrity of Brain Cyst Isolated from Chronically Infected Mice.
[0417] Examination of brains of mice infected with Δbcla mutants demonstrated that cyst formation could still occur in the context of the mutated strains (
[0418] We then assessed whether the surface deformities would make the cyst fragile, a phenotype previously reported for brain Δcst1-containing cysts (Tomita et al., 2013). While during their isolation cysts were subjected to mechanical stress to release them from brain tissue to purify them by isopycnic centrifugation (see method), we did not observe during this harsh procedure that Δbcla-containing cysts were more fragile than the WT cysts (data not shown), yet few of them broke apart regardless of the genetic background.
[0419] The deletion of BCLA does not impair either the wall staining by Dolichos bifluorus lectin (DBA) of cysts isolated from the brain of chronically infected mice (data not shown). Therefore and as already concluded on tachyzoites treated by FR235222, BCLA is not directly implied in the GalNAc glycosylation of the cyst wall. The viability of bradyzoite within the cysts is conditioned to the permeability of the wall to the nutrients that come from the host cell, yet the latter is very limited, the wall functioning as a sieve, to avoid the components of the immune response. To test if the wall permeability was altered to some way in absence of BCLA we monitored the entry into the cysts of fluorophores, typified by different sizes ranging from 3 to 40 kDa. Only intact cysts (with no leak of parasites) were visualized under the microscope and examined. The permeability was quite similar between WT and BCLA-deficient cysts with either the 3-kDa (diffuse pattern throughout the cyst matrix) or the 10-kDa (diffuse pattern with punctuated location) dyes. Interestingly, the fluorescent tracer with higher molecular mass (40-kDa) was not efficiently able to cross the cyst wall as reported previously (Lemgruber et al., 2011). Moreover, the weak labelling was even different between the strains, likely because Δbcla-containing cysts are more “loosed” and permeable than those containing the parental strain, more filled with bradyzoites surrounded by a less permeable, well defined and continuous cyst wall (data not shown). Overall our results show that BCLA is dispensable for proper cyst function in vivo yet the protein has an architectural role in the cyst wall that may lead to a cyst wall permeability defect phenotype.
[0420] BCLA is not Essential for Efficient Oral Infection by Toxoplasma Bradyzoite-Containing Cysts.
[0421] To examine the functional consequences in vivo of BCLA-dependent cysts deformation, we fed mice with Δbcla- or parental strains-containing cysts and assessed the virulence and infectivity in two different mice genetic backgrounds. C57BL/6 mice were orally infected with 46 cysts of the 76k-GFP-luc-Δbcla or 76k-GFP-WT strains and the kinetics of invasion and dissemination of the parasite in the gut as well as the local immune responses elicited by the parasite were studied. At day 8 of infection, the levels of T. gondii-specific IgG in mouse sera were quite similar (data not shown) and they were no significant differences in the parasite load in ilea (
[0422] High-Level Expression and Purification of BCLA Chimeric Peptides for Serological Diagnosis
[0423] The humoral and cellular defences of the innate immune system are the body's first lines of defense against T. gondii. Antibodies were reported to assist the clearing of parasites during acute infection and mediate resistance to secondary Toxoplasma infection (Sayles et al., 2000). As such, once immunity has been established, IgG protects the fetus from a vertical transmission during pregnancy. While serologic differentiation between acute and chronic infections has clinical and epidemiological relevance, yet there is currently no bradyzoite-specific serological assay for toxoplasmosis to estimate accurately the time of infection as well as the presence of cysts. Moreover, since reactivation can both occur in perfectly immunocompetent patient (e.g. retinochoroiditis) and in immunocompromised patients, and cyst presence in brain was recently suspected to be linked to some neuropsychiatric disorders. Thus, detecting toxoplasmic antibodies directed against semi-dormant cysts would be a significant improvement to the serological diagnosis of toxoplasmosis by opening new diagnostic perspectives. However, few components of the cyst wall or surface bradyzoite have been identified, and none were shown to serve as antigen for serology purpose, at least in commercial kits. The ideal antigen should be expressed exclusively in latent bradyzoite stage and ideally should be exposed to the surface of the cyst, two features found in BCLA that motivated us to test its antigenicity.
[0424] In order to obtain highly pure and abundant quantities of BCLA required for sera WB testing, we opted to recombinantly express the C-terminal domain end of BCLA (res 1100 to 1275, hereafter referred as rBCLA) which is predicted as structured, as opposed to the rest of the protein containing core repeated motifs (
[0425] rBCLA does not React with Sera of Acutely Infected Mice but Constitutes an Excellent Antigen for the Detection of Anti-T. gondii IgG from Chronically Infected Mice
[0426] We first performed immunoblots on sera collected from mice in acute phase of infection. rBCLA protein is apparently not reacting with sera of mice acutely infected by atypical (COUG, haplotype 11), virulent (RH, type I) or cystogenic (76K, type II) strains (
[0427] rBCLA is Exclusively Detected in Sera of Mice Persistently Infected by Cystogenic Strains
[0428] rBCLA was shown to have specific reactivity for cystogenic strains prone to make latent infections (
[0429] Limited Proteolysis to Find Antigenic Sub-Fragments within rBCLA
[0430] With the objective to recover a highly antigenic sub-fragment of rBCLA, a limited proteolysis on the purified sample was undertaken using trypsin, chymotrypsin, elastase and papain. Analysis of the proteolysis reaction by SDS PAGE (
[0431] rBCLA Also Reacts with Human Sera, Yet the Pattern of Positivity is Still Under Investigation
[0432] We next showed that mice infected with a positive amniotic fluid from a pregnant woman primary infected during pregnancy with proof of congenital toxoplasmosis are clearly reacting with rBCLA, contrary to those infected with qPCR negative amniotic fluid or placentas (
TABLE-US-00017 TABLE 2 Toxoplasma strains used in the present invention Strain Genotype Reference or source RH Type I Lab strain RHΔku80 RH Δku80 (Huynh and type I strain Carruthers, 2009) GT1 SNF.sup.R Type I (Behnke MS et al. 2011) PruΔku80 Prugniaud Δku80 (Fox et al., 2011) type II strain PruΔku80Δbcla Type II Genetically modified from (Fox et al., 2011) ME49 FUDR.sup.R Type II (Behnke MS et al. 2011) 76K-GFP-Luc 76K type II strain ex- Gift from Michael pressing ectopically Grigg (NIH) GFP and Luciferase type II strain 76K-GFP-Luc- Type II Genetically modified Δbcla from (NIH) CTG Type III (Rosowski EE, et al. 2010)
TABLE-US-00018 TABLE 3 rBCLA antigen is reacting with some sera and aqueous humours from humans Age IgG IgG IgM IgM Serological and rBCLA ID Sex (years) Sample Medical context Architect ® Vidas ® Architect ® Vidas ® clinical status status α 1 M 89 Serum Corneal 32.3 237.0 0.04 0.04 Past immunity, +++ transplant ocular toxoplasmosis (reactivation)? 2 M 83 Serum Ocular >2000.0 >300.0 0.15 0.11 Past immunity, +++ toxoplasmosis ocular toxoplasmosis (reactivation) 3 M 83 Aqueous Ocular Aqueous humor with Ocular +++ humor toxoplasmosis neosynthetized Ab & PCR+ toxoplasmosis (reactivation) 4 F 85 Serum Ophthalmological 24.3 73.0 0.57 0.22 Past immunity, ++ disease residual IgM, ocular toxoplasmosis (reactivation) ? β 5 F 57 Serum Hematology 46.0- >300.0 0.07 0.06 Toxoplasmosis + (ID) reactivation? 6 F 28 Serum Reactivation of 19.7 26.0 4.93 6.05 Toxoplasmosis + toxoplasmosis, reactivation hematological disease 7 F 68 Serum Toxoplasmosis >2000.0 >300.0 2.7 4.11 Toxoplasmosis ++ reactivation, reactivation hematological disease γ 8 F 26 Serum Seroconversion 431.8 >300.0 2.65 1.95 Recent ++ during seroconversion pregnancy (infection ~1 month ago) 9 F 20 Serum Seroconversion 12.7 12.0 1.05 1.06 Recent ++ during seroconversion pregnancy (infection ~1 month ago) 10 F 21 Serum Seroconversion 32.0 19.0 1.56 1.26 Recent + during seroconversion pregnancy (infection ~1.5 month ago) δ 11 M 2 months Serum Congenital 168.3 150.0 12.30 6.45 Congenital ++ toxoplasmosis toxoplasmosis ε 12 M 55 Serum Pre-graft 29.9 174.0 0.30 0.37 Past immunity — monitoring (renal transplant) 13 F 24 Serum Hematological 43.6 100.0 0.15 0.30 Past immunity — disease (without reactivation) 14 F 33 Serum Seroconversion 45.9 30.0 2.41 1.96 Recent — during seroconversion pregnancy (infection ~2.5 months ago) 15 F 71 Serum Cornea guttata 4.9 16 0.08 0.06 Past immunity — (no ocular toxoplasmosis) ζ 16 F 30 Serum Healthy 0.15 / 0.04 / Seronegative — 17 F 61 Serum Hematological 0.20 / 0.09 / Seronegative — disease 18 M 6 months Serum Serological 1.60 1.00 0.06 0.03 Seronegative — follow-up of an infant suspected of congenital toxoplasmosis 19 M 64 Serum HIV patient 0.20 / 0.12 / Seronegative — 20 F 34 Serum Pregnant 0.10 / 0.04 / Seronegative — woman (serological follow-up during pregnancy)
[0433] Cutoffs recommended by manufacturers for interpretation of serologic values using Vidas® and Architect®
[0434] Vidas® IgG (IU/mL): negative<4; grayzone: 4.0≤x<8.0; positive: ≥8.0
[0435] Vidas® IgM (index): negative<0.55; grayzone: 0.55≤x<0.65; positive: ≥0.65
[0436] Architect® IgG (IU/mL): negative<1.6; grayzone: 1.6≤x<3.0; positive: ≤3.0
[0437] Architect® IgM (index): negative<0.50; grayzone: 0.50≤x<0.60; positive: ≥0.60
[0438] Epitope Mapping in rBCLA Positive Patients Reveals a Multitude of Antigenic Regions within rBCLA and Consistent Reactivity within the Repeated Region.
[0439] With the specific immunogenic quality of rBCLA being proven by western blot in a series of sera from different clinical categories. One of the main objectives was to develop an ELISA based assay to screen larger serum cohorts in a cost-effective, robust and fast manner. However, to correctly setup such an assay, which could be almost entirely based on chemically synthetized peptides, a more precise understanding of the local epitope immunogenicity of BCLA was required. To do so, we designed and synthetized cellulose printed peptide arrays covering both the repeated region and rBCLA domain (
[0440] ELISA Titration Using rBCLA and Repetition Peptides Demonstrates that BCLA Seropositivity is Higher within Acute and Chronically Infected Individuals.
[0441] Upon establishing stringent rules to categorise and differentiate different clinical profiles. 123 sera (all taken from different individuals) were tested with the developed BCLA-ELISA test. Their ELISA score, expressed in international units (UI) is displayed (
[0442] 1) “Seronegative”, which regroups all patients (healthy or with other pre-conditions) with SAG1 IgG/IgM negative serologies.
[0443] 2) “Past immunity”, which regroups all patients (healthy or with other conditions), which are categorised as SAG1 positive IgG but with no SAG1 reactive IgM and which do not fall into the next three categories.
[0444] 3) “Active toxoplasmosis in immunocompromised patient”, which regroups all SAG1 IgG positive and immunocompromised patients with a proven symptomatic toxoplasmosis (regrouping disseminated, cerebral and primary toxoplasmosis).
[0445] 4) “Asymptomatic□ serological reactivation in immunocompromised patients”, which regroups all patients immunocompromised undergoing a serological reactivation but without visible symptoms.
[0446] 5) “Ocular toxoplasmosis”, which regroups patients with SAG1 positive serologies and suffering from a proven ocular toxoplasmosis.
[0447] From this analysis several observations can be made. First, all groups when compared to the seronegative group display a significant increase in median BCLA titrations and have much higher positivity rates. This proves in humans, the direct correlation between a SAG1 seropositivity status and the ability to develop a BCLA positivity status. This also shows a current discrepancy between SAG1 negative serologies and BCLA serologies which still display a false positive discovery rate of around 10%. This can be explained in some sera by non-specific interactions with the different BCLA epitopes, strongly immunogenic exogenous bacterial contaminants co-purified with rBCLA and potentially truly BCLA positive patients with negative SAG1 serologies. The second main observation is that some clinical profiles have a tendency to generate vastly stronger immunogenic reactions, most notably the “Asymptomatic□ serological reactivation in immunocompromised patients” group where BCLA serologies are titrated well beyond the median positive BCLA serology in the “past immunity” group. The final observation is that for some groups where a BCLA positivity should always be expected, such as in the case of “Active toxoplasmosis in immunocompromised patient” and “ocular toxoplasmosis”, a minority of serologies remain negative or under the positivity cut-off. This observation can highlight a lack in sensitivity from the ELISA test or potentially illustrates the fact that BCLA serologies can become negative during immunosuppression.
[0448] The ELISA Test is Also Consistent in Linking Positive BCLA Serologies in Mice to a Proportional Cyst Burden.
[0449] Overall, the semi-quantitative analysis of anti-rBCLA antibody titers identified BALB/c and NMRI mice likely bearing WT cysts as highly responsive to BCLA with an increased yield over time (
Discussion
[0450] Infections by Toxoplasma gondii lead to an acute systemic phase by which the zoites rapidly establish themselves and further complete their developmental program as bradyzoites enclosed in cysts surrounded by a thick cyst wall that persist in brain, heart and skeletal muscle (Jeffers et al., 2018). The host immune response is promptly able to control the tachyzoite population expansion, leading to a life-long immunity typified by seroconversion. Yet, since the developmental transitions from tachyzoite to bradyzoite are fully bidirectional any impairment of immune functions (e.g. AIDS patients, haematological diseases and immunosuppressive treatments) can result in reactivation of latent infection that may cause encephalitis and focal brain lesions, pulmonary or disseminated disease.
[0451] The diagnosis of acute and chronic toxoplasmosis in immunocompetent subjects relies mainly on serology, and because infections are often asymptomatic, serologic diagnosis is in many cases retrospective, as it is based on the demonstration of seroconversion, e.g. during pregnancy or in a graft setting (Robert-Gangneux and Darde, 2012). Increased levels of IgM and IgA antibodies are serologic indicators of primary/acute infections and, while a high IgG-avidity rules out a primary infection, persistent and steady-state IgG levels in absence of IgM typifies latent infections (Dard et al., 2016). However, the interpretation of the serological results remains difficult, even for well-trained specialists. The current challenges to overcome are: (i) to discriminate between recent and more distant infections; (ii) to diagnose congenital toxoplasmosis in infants and reactivation in immunocompromised patients; and iii) to establish the origin of infection, i.e. oocysts versus cysts. While many methods have been developed in the last decades to improve the accuracy and sensitivity of serological assays, they poorly address the aforementioned concerns. An obvious reason is that many, if not all, commercial serologic test kits are detecting lysate or recombinant antigens that are prevalently expressed at the tachyzoite stage (e.g. SAG1) or common to both the infectious stages of the parasite (e.g. GRA8).
[0452] Currently, while there is no reliable bradyzoite-specific serological assay for toxoplasmosis to estimate the sources of infection worldwide nor to discriminate accurately between acute and latent infections, progress has been made. Indeed, recent proteomic studies have shed light on the repertoire of sporozoite-specific proteins (Fritz et al., 2012; Possenti et al., 2013), which has revealed CCp5A as a serological marker able to differentiate the parasite stage that infected chickens, pigs and mice, with specific reactivity for oocyst-infected animals (Santana et al., 2015).
[0453] However and despite early studies reporting that specific bradyzoite antigens including BAG1 contribute to the stimulation of both humoral (Mun et al., 1999) and cell-mediated immunity against T. gondii infection (Di Cristina et al., 2004), no bradyzoite/cyst antigens are currently considered as potential markers of latent infection in diagnostic tests. The search for bradyzoite/cyst-specific markers has been somewhat limited by the ability to harvest sufficient mouse brain cysts to analyze the specific proteome of the latent stage. In this study, we found a way to circumvent this problem by de-repressing bradyzoite genes in cell culture while manipulating the chromatin state of tachyzoite with epi-drugs. Hundred of bradyzoite-restricted proteins were therefore identified, including BCLA.
[0454] The protein BCLA was shown to be not essential to initiate or sustain latent infections, yet BCLA-deficiency resulted in a quite singular phenotype typified by the deformation and loss of circularity of cerebral cysts in murine model. So far, two cyst wall-associated proteins, i.e. BPK1 and CST1, were involved in the structural integrity of T. gondii cysts (Jeffers et al., 2018). In Δbpk1 strain cysts are smaller and more sensitive to pepsin-acid treatment and unlike BCLA, Δbpk1 strain has reduced ability to cause oral infection (Buchholz et al., 2013). CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall (Tomita et al., 2013). A defect of glycosylation may also explain the deformation of Δbcla cysts. Indeed, we have preliminary interactome data showing BCLA co-purified with a Jacaline-binding protein (data not shown), which is a lectin binding to GalNAcα1-Ser/Thr oligosaccharide, that covers bradyzoites-surrounding PVM (Tomita et al., 2017). Further investigation is needed to establish whether this interaction is responsible for the BCLA deficiency-mediated peculiar phenotypes.
[0455] Having no clear BCLA-associated phenotype in mice regardless of the route and the time of infection, we oriented our investigations on the propensity of BCLA to play an immunogenic role. We therefore reach another milestone by producing rBCLA as a recombinant protein with a high degree of purity which offers the opportunity to standardize the serological tests and to some extent to reduce the manufacturing costs, in case this antigen turns out to be interesting for serology. In fact, we brought strong data supporting that rBCLA is antigenic and constitutes an excellent antigen candidate for the detection of anti-T. gondii IgG in chronically infected mice. Strikingly, we clearly correlated the strong detection in sera of the antigen rBCLA and the cyst burden in the brain of all mice latently infected by type II cystogenic strains. A similar study claims that MAGI antibody level correlates with brain cyst burden but their experimental settings are somewhat biased by the use of an irrelevant model of chronic type I (GT1) infection that requires anti-T. gondii chemotherapy to control the proliferation of tachyzoites during the acute stage and to avoid animal death (Xiao et al., 2016).
[0456] Remarkably, rBCLA did not react with IgM nor IgA (data not shown), markers frequently associated with acute infection, but only with IgG and exclusively in subchronic infections. This result clearly contrasts with the observation that tissue cyst fed mice showed significant IgM response at day 10 (Döşkaya et al., 2018) and reinforces the idea of a humoral response against BCLA during the latent stage of infection. Likewise, mice inoculated by oral gavage with tissue cysts did not produce antibodies directed against BCLA at the time of acute infection (
[0457] Finally, anti-rBCLA antibodies have been detected in some human sera from patients with ocular toxoplasmosis following toxoplasmic reactivation, during toxoplasmosis reactivation linked to immunosuppression or congenital toxoplasmosis. These findings are in agreement with the conclusions drawn from the murine model that rBCLA makes a fantastic serological marker of the presence of tissue cysts in the chronically infected hosts.
EXAMPLE 2 (VHH PRODUCTION)
[0458] Immunization
[0459] Llamas SEL005 and SEL006 were immunized via Eurogentec via 4 injections at day 0, 14, 28 and 35. Sera was obtained at day 0, day 28 and day 43. Peripheral blood mononuclear cells (PBMC) were obtained from a large bleed at day 43.
[0460] Immune Response
[0461] The immune response of SEL005 and SEL006 was tested by assessing the presence of rBCLA-specific antibodies in sera of day 43. A MaxiSorp plate was coated with 200 ng antigen per well overnight at 4° C. After three times washing with PBS containing 0.05% Tween-20 the plate was blocked with 4% milk powder in PBS (MPBS). Next, a serial dilution of the sera in 1% MPBS was added to the wells and incubated for 1 hour. Unbound antibodies were removed during washing with PBS-Tween. Subsequently, bound antibodies were detected with rabbit-anti-VHH (clone K1216) and donkey-anti-rabbit coupled to HRP. Antibody binding was quantified by the colorimetric reaction of 0-phenylenediamine (OPD) in the presence of H2O2 at 490 nm. Llamas SEL005 and SEL006 show a very good response against His rBCLA.
[0462] Library Construction of SEL005 Day 43 and SEL006 Day 43
[0463] RNA Isolation and cDNA Synthesis
[0464] Peripheral blood lymphocytes were isolated from a large bleed at day 43 from which RNA was isolated at Eurogentec. Precipitated RNA was dissolved in RNase-free MQ and the RNA concentrations were measured. To assess the quality of the RNA, 5 μl of the dissolved RNA was analyzed on gel.
[0465] About 40 μg RNA (4 reactions of 10 μg each) was transcribed into cDNA using a reverse transcriptase Kit (Thermo Fisher Scientific). The cDNA was purified on Macherey Nagel PCR clean-up columns. Variable domains of the heavy chains (both conventional and heavy chain-only) fragments were amplified using primers annealing at the leader sequence region and at the CH2 region. 5 μl was loaded onto a 1% TBE agarose gel for a control of the amplification.
[0466] After this control, the remaining of the sample was loaded on a 1% TAE agarose gel and the 700 bp fragment was excised and purified from the gel. A total of 80 ng of isolated PCR product was used as a template for the nested PCR (end volume 800 μl) to introduce SfiI and Eco91I restriction sites to either end of the VHH gene. The amplified VHH fragment was cleaned on Macherey Nagel PCR cleaning columns and eluted in 120 μl. The eluted DNA was first digested with SfiI, followed by Eco91I. As a control of the restriction digestion, 4 μl of this mixture was loaded onto a 1.5% TBE agarose gel.
[0467] After the restriction digestion, the samples were loaded on a 1.5% TAE agarose gel. The 400 bp fragment was excised from the gel and purified on Machery Nagel gel extraction columns. The purified 400 bp VHH fragments (˜330 ng) were ligated into the pUR8100 phagemid vector (˜1 μg) and transformed into TG1 E. coli.
[0468] Library Size
[0469] The transformed TG1 were titrated using 10-fold dilutions. 5 μl of the dilutions were spotted on LB-agar plates supplemented with 100 μg/ml ampicillin and 2% glucose. The number of transformants was calculated from the spotted dilutions of the transformed TG1 culture (keeping in mind that the final volume of the transformation is 8 ml). The total number of transformants and thereby the size of the library was calculated by counting colonies in the highest dilution and using the formula below:
Library size=(amount of colonies)*(dilution)*8 (ml)/0.005 (ml; spotted volume)
[0470] The VHH insert frequency in the phagemid vector was determined by picking 24 different clones and performing a colony PCR. Bands of ˜700 bp indicate a successfully cloned VHH fragment. Bands of ˜300 bp indicate an empty plasmid. The insert frequency for library SEL005 day 43 is 100%. For library SEL006 day 43 the insert frequency is almost 95% (
[0471] Phage Production and Selection
[0472] Phages were produced from the library as outlined below: E. coli TG1 containing libraries SEL005 day 43 and SEL006 day 43 were diluted from the glycerol stock up to an OD600 of 0.05 in 2×YT medium containing 2% glucose and 100 μg/ml ampicillin. The number of bacteria in this inoculum was at least 10× the library size (>109 bacteria in the inoculum). This culture was grown at 37° C. for 2 hours to reach an OD600 of ˜0.5. Subsequently, about 7 ml of the culture was infected with helper phage VCS M13 using a MOI (multiplicity of infection) of 100 for 30 minutes standing at 37° C. Infected bacteria were spun down and resuspended into 50 ml fresh 2×YT medium supplemented with both ampicillin (100 μg-/ml, for the phagemid) and kanamycin (25 μg/ml, for the M13 phage) and grown overnight at 37° C., shaking. Produced phages were precipitated from the supernatant of the cultures using PEG-NaCl precipitation. Titer of the produced phages was calculated by serial dilution of the phage and infection of E. coli TG1. Titer of the produced phages were 3×1011/ml for SEL005 day 43 and 6×1011/ml for SEL006 day 43, respectively, which was sufficient for continuing with the selections.
[0473] For the 1st round of panning/selections, 20 μl of the precipitated phages (˜1010 phages, which is >100-fold the diversity of the libraries) were applied to wells coated with His rBCLA. In short, 100 μl antigen was coated on the MaxiSorp overnight at 2 concentrations 5 μg/ml and 0.5 μg/ml. As a negative control, one well was incubated with PBS only. Next day after removal of non-bound antigen, the plate was washed three times with PBS and blocked with 4% milk powder in PBS (MPBS). At the same time freshly precipitated phages were pre-blocked with 2% MPBS for 30 minutes. Pre-blocked phages were incubated with directly coated His rBCLA for 2 hours. Upon extensive washing with PBS-Tween and PBS, bound phages were eluted with 0.1M TEA-solution, which was subsequently neutralized with 1M Tris/HCl pH7.5. Eluted phages were serially diluted and then used to infect TG1 bacteria and spotting on LB-agar plates supplemented with 2% glucose and 100 μg/ml ampicillin and incubated at 37° C.
[0474] For the 2nd round of selection, new phages were produced of rescued output from the selection on 5 μg/ml His rBCLA (highest concentration). The overnight grown rescued outputs were diluted 100-fold in 5 ml fresh 2×YT medium supplemented with 2% glucose and 100 μg/ml ampicillin and grown for 2 hours until log-phase. Subsequently 1 μl of helper phage VCS M13 was added and incubated at 37° C. for 30 minutes. Cultures were allowed to produce phages overnight at 37° C. Produced phages were precipitated from the supernatant of the cultures using PEG-NaCl precipitation.
[0475] Subsequently, for the 2nd round of panning/selection, 1 μl of the precipitated phages was applied to wells coated with His rBCLA as indicated below: antigen was coated on the MaxiSorp plate overnight at 3 concentrations (5 μg/ml, 0.5 μg/ml and 0.05 μg/ml). As a negative control, one well was incubated with PBS only. Next day, after removal of non-bound antigen, the plate was washed three times with PBS and blocked with 4% MPBS. At the same time freshly precipitated phages were pre-blocked in 2% MPBS for 30 minutes as described above. Pre-blocked phages were incubated with directly coated His rBCLA for 2 hours. Upon extensive washing with PBS-Tween and PBS, bound phages were eluted with 0.1M TEA-solution and subsequently neutralized with 1M Tris/HCl pH7.5. Eluted phages were serially diluted and then used to infect TG1 cells and spotting on LB-agar plates supplemented with 2% glucose and 100 μg/ml ampicillin and incubated overnight at 37° C.
[0476] Screening after 2 Rounds of Phage Display Selections
[0477] Rescued outputs of the 2nd round of selection on His rBCLA were plated out in order to pick single clones. For master plate ERB-1, a total of 92 single clones were picked in a 96-wells plate.
[0478] In order to screen master plate ERB-1 for His rBCLA-binders, periplasmic extracts containing monoclonal VHH were produced. The master plate was cultivated at 37° C. in 2×YT medium supplemented with 2% glucose and 100 μg/ml ampicillin and stored at −80° C. after addition of glycerol to a final concentration of 20%. For the production of periplasmic extracts, master plate ERB-1 was duplicated into a deep well plate containing 1 ml 2×YT medium supplemented with 0.1% glucose and 100 μg/ml ampicillin and grown for 3 hours at 37° C. before adding 1 mM IPTG for induction of VHH expression. The VHH expression was conducted overnight at room temperature. Periplasmic extracts were prepared by collecting the bacteria by centrifugation, resuspension of this pellet into 120 μl PBS and one freeze-thaw cycle. Bacteria were centrifuged to separate the soluble periplasmic fraction containing the VHH from the cell debris (pellet). To test the binding specificity of the monoclonal VHH by ELISA, His rBCLA (100 ng/well in PBS) was coated overnight onto a MaxiSorp plate at 4° C. The coated plate was washed and subsequently blocked using 4% MPBS. The blocked wells were incubated with 10 μl of the periplasmic extracts and 40 μl 1% MPBS for 1 hour at room temperature. Unbound VHH were removed by washing with PBS containing 0.05% Tween-20. Subsequently, bound VHH were detected with rabbit-anti-VHH (clone K976) and donkey-anti-rabbit coupled to HRP. Binding of the VHH was quantified by the colorimetric reaction of OPD in the presence of H2O2 at 490 nm. All clones of master plate ERB-1 were able to bind specifically to His rBCLA. There is no difference shown between the two libraries used.
[0479] Sequence Analysis of his rBCLA Binding VHH
[0480] Based on the ELISA results, 17 clones (ERB-1A1, ERB-1F1, ERB-1A2, ERB-1E2, ERB-1F2, ERB-1G2, ERB-1B3, ERB-1H4, ERB-1A5, ERB-1G6, ERB-1D7, ERB-1F7, ERB-1G8, ERB-1E9, ERB-1E10, ERB-1B11 and ERB-1A12) were selected for sequence determination. These clones were picked based on binding in ELISA and should represent the majority of the clones selected from the different output
[0481] Cloning and Production of VHH Selected on his rBCLA
[0482] From all the sequenced clones, 7 clones (ERB-1F1, ERB-1F2, ERB-1H4, ERB-1G6, ERB-1D7, ERB-1B11 and ERB-1A12) were chosen as a good representative of the found VHH sequences. These VHH were then subcloned from the phagemid vector into the expression vector pMEK222 using SfiI and Eco91I restriction enzymes. Recloning into pMEK222 also adds a FLAG and His-tag to the C-terminus of the VHH, allowing detection and affinity purification. For the production, pre-cultures were prepared by growing the bacteria containing the plasmids with the selected VHH in 8 ml 2×YT medium supplemented with 2% glucose and 100μ/ml ampicillin overnight at 37° C. The pre-cultures were diluted into 800 ml fresh 2×YT that was pre-warmed at 37° C. and supplemented with 100 μg/ml ampicillin and 0.1% glucose. The bacteria were grown for 2 hours at 37° C. before induction of the VHH expression with 1 mM IPTG. The VHH were expressed for 4 hours at 37° C. and bacteria were harvested by centrifugation. Bacteria pellets were resuspended into 30 ml PBS and frozen at −20° C.
[0483] Purification and Analysis of the VHH
[0484] Frozen bacteria pellets were thawed at room temperature and cell debris was spun down by centrifugation. VHH were purified from the supernatant (soluble fraction) using affinity of the His-tag to Cobalt charged sepharose beads (Immobilized Metal Affinity Chromatography (IMAC) using TALON beads). Bound VHH were eluted with 150 mM imidazole and dialyzed against PBS.
[0485] The protein concentration was measured using absorption at 280 nm and corrected according the molar extinction coefficient and the molecular weight of the different VHH.
[0486] As a quality check, 1 μg of purified VHH was loaded on a SDS-PAGE.
[0487] The binding of purified VHH to immobilized His rBCLA was analyzed by ELISA. A MaxiSorp plate was coated with 200 ng/well antigen overnight at 4° C. in PBS. After blocking the wells with 4% MPBS, a serial dilution of the VHH was added to the coated wells and incubated for 1 hour at room temperature. After washing unbound VHH, bound VHH were detected using a mouse-anti-flag (clone M2) and donkey-anti-mouse coupled to HRP. Binding was quantified by measuring colorimetric reaction of OPD+H2O2 at 490 nm. ERB-1G6, ERB-1B11 and ERB-1A12 show a subnanomolar apparent affinity to immobilized His rBCLA. ERB-1F1 and ERB-1F2 show a low nanomolar affinity. ERB-1H4 and ERB-1D7 show a molar apparent affinity to His rBCLA.
CONCLUSIONS
[0488] Immunization of the llamas SEL005 and SEL006 resulted in a good immune response. The generated libraries were of a good size and insert frequency. Phage display selections on His rBCLA has resulted in a number of good clones of which 3 (ERB-1G6, ERB-1B11 and ERB-1A12) show a very good apparent affinity of which ERB-1G6 also shows a high production level in E. coli.
TABLE-US-00019 TABLE 4 Useful amino acid sequences for practicing the invention SEQ ID NO Nucleotide or amino acid sequence 1: full lenght polypeptide MKLFFKLVLAGVSSIFAAQCLAGAVAARAGMPEITI BCLA REEEEELFPSLDDVLDTSPFPARLWMGPGEKQATES HTPATIPTAYKSTPGLSATVTGGEDGSAKMVGMDV AKKPVKVSVKKEEENKDVEANEDGWDYIVSKGVP GKIPATVMDEARKADVVADGEAKPAMREAQERRK PWETQEEKILVLPKVQRILALPKEEKKHVSTAKGEE PFSSKEEERHVLLNGEERKPVVPRAGREQPAVPRQE EQKLVLQKTERKPVLPEEDQKPVLPETGAKHVLPEI ATKSTLTQKEVTKPVETRQDMRGTAGSMEEKKPVL PGEGKRHVLPKDETKPALTEEKRTKPVEPRKEMESP ARPMEEEKPVLPGEGERHVLPKDERKPALTDEKRT KPGGPRTEMERPAAGSMEKDKLVLPGEGEGHVLPK HETKPALTDEKRTKPGGPRTEMERPAAGSMEKEKP VLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTEM ERPAAGSMEKDKLVLPGEGEGHVLPKHETKPALTD EKRTKPGGPRTEMERPAAGSMEKDKLVLPGEGEGH VLPKHETKPALTDEKRTKPGGPRTEMERPAAGSME KDKLVLPGEGEGHVLPKHETKPALTEEGRTEPIEPR KAMERPAGAMEKTKPVLPGEGERHVLPKAETKTA LTEEERTEPGGPRMAMERPAAGSMEKKKPVSPGEG EGHVLPKHETKPALTDEKRTKPGGPRTEMERPAAG SMEKDKLVLPGEGERHVLPKHERKPALTDEKRTKP GGPPTEMERPAAGSMEKDKLVLPGEGERHVLPKDE TKPALTEEKRTKPGGPRTEMERPAAGSMEKEKPVL PGEGERHASPKDEMKPALTDEKRTKPGGPRKEMER PAAGSMEKEKPVLPGEGERHVLPKDEQKAALTQKE VTNPVEPRKEMERPAAPIEGEKGVVSSEEEKPVSPK EATRRILPKEGKESLGTRKEEVKPIVRRAKRGRRIA QKGKEKQIAPKEGKKPAVPKEGEERPAEPTEGEERP VGPKEGEERPVGPKEGEERPVVPDVDKEKPVVPEG DKEKPVVPEGDKDHPALPEQDEEKHATWEKEMIPG VGDKTEASVLDSIENAVQKVLENLLKAAAGELQPA EAEEARLLVADLKAVVDTAEQVRVEGEAFFRASVD LYEAVKNLRDSEEKLRPLTKGELVDVVRQFLATQIF VQDRASAFLRVFERLAELLAAEQMKAVFAMVEEG VSSSERVARVAGELVPMMKKDRERRYGDLVAVTS WFMRRMEHI 2: rBCLA MIPGVGDKTEASVLDSIENAVQKVLENLLKAAAGE LQPAEAEEARLLVADLKAVVDTAEQVRVEGEAFFR ASVDLYEAVKNLRDSEEKLRPLTKGELVDVVRQFL ATQIFVQDRASAFLRVFERLAELLAAEQMKAVFAM VEEGVSSSERVARVAGELVPMMKKDRERRYGDLV AVTSWFMRRMEHI 3: HisTag-rBCLA MGHHHHHHHHENLYFQGMIPGVGDKTEASVLDSI ENAVQKVLENLLKAAAGELQPAEAEEARLLVADL KAVVDTAEQVRVEGEAFFRASVDLYEAVKNLRDS EEKLRPLTKGELVDVVRQFLATQIFVQDRASAFLRV FERLAELLAAEQMKAVFAMVEEGVSSSERVARVA GELVPMMKKDRERRYGDLVAVTSWFMRRMEHI 4. TgR1 MRGTAGSMEEKKPVLPGEGKRHVLPKDETKPALT EEKRTKPVEPRKE 5. TgR2 MESPARPMEEEKPVLPGEGERHVLPKDERKPALTD EKRTKPGGPRTE 6: TgR3 MERPAAGSMEKDKLVLPGEGEGHVLPKHETKPAL TDEKRTKPGGPRTE 7. TgR4 MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALT DEKRTKPGGPRTE 8. TgR5 MERPAAGSMEKDKLVLPGEGEGHVLPKHETKPAL TDEKRTKPGGPRTE 9. TgR6 MERPAAGSMEKDKLVLPGEGEGHVLPKHETKPAL TDEKRTKPGGPRTE 10. TgR7 MERPAAGSMEKDKLVLPGEGEGHVLPKHETKPAL TEEGRTEPIEPRKA 11. TgR8 MERPAGAMEKTKPVLPGEGERHVLPKAETKTALT EEERTEPGGPRMA 12. TgR9 MERPAAGSMEKKKPVSPGEGEGHVLPKHETKPAL TDEKRTKPGGPRTE 13. TgR10 MERPAAGSMEKDKLVLPGEGERHVLPKHERKPAL TDEKRTKPGGPPTE 14. TgR11 MERPAAGSMEKDKLVLPGEGERHVLPKDETKPAL TEEKRTKPGGPRTE 15. TgR12 MERPAAGSMEKEKPVLPGEGERHASPKDEMKPAL TDEKRTKPGGPRKE 16. TgR13 MERPAAGSMEKEKPVLPGEGERHVLPKDEQKAAL TQKEVTNPVEPRKE 17. Peptide 1 of TgR1 and EMRGTAGSMEE TgR11 18. Peptide 2 of TgR1 VLPKDETKPALT 19. Peptide 1 of TgR2 EMESPARPMEE 20. Peptide 2 of TgR2 VLPKDERKPALT 21. Peptide 1 of TgR3 to EMERPAAGSMEK TgR7 and of TgR9 to TgR13 22. Peptide 2 of TgR3 to VLPKHETKPALT TgR7 and TgR9 23. Peptide 1 of TgR8 EMERPAGAMEK 24. Peptide 2 of TgR8 VLPKAETKTALT 25. Peptide 2 of TgR10 VLPKHERKPALT 26. Peptide 2 of TgR12 ASPKDEMKPALT 27. Peptide 2 of TgR13 VLPKDEQKAALT 28. primer TgBCLA-KO- aagttgatcactattcgtgaagaagg CRISP-FWD 29. primer TgBCLA-KO- aaaaccttcttcacgaatagtgatca CRISP-REV 30. primer TgBCLA-HF- aagttggaacggcggtacggcgaccg CRISP-FWD 31. primer TgBCLA-HF- aaaacggtcgccgtaccgccgttcca CRISP-REV 32. domain A of rBCLA GELQPAEAEEARLLVADLKAV 33. domain B of rBCLA VRVEGEAFFRASVDLYEA 34 domain C of rBCLA KLRPLTKGELVDVVRQ 35. peptide 36 of rBCLA TQIFVQDRASAFLRV 36. peptide 44 of rBCLA AAEQMKAVFAMVEEG 37. peptide 12 of rBCLA GELQPAEAEEARLLV 38. peptide 13 of rBCLA QPAEAEEARLLVADL 39. peptide 14 of rBCLA EAEEARLLVADLKAV 40. peptide 21 of rBCLA VRVEGEAFFRASVDL 41. peptide 22 of rBCLA EGEAFFRASVDLYEA 42. peptide 23 of rBCLA AFFRASVDLYEAVKN 43. peptide 30 of rBCLA KLRPLTKGELVDVVR 44. domain A of TgR4 AAGSMEKEKPVLPGEGEGH 45. domain B of TgR4 VLPKHETKPALTDEKRTKPGGP 46. peptide 3 of TgR4 AAGSMEKEKPVLPGE 47. peptide 4 of TgR4 GSMEKEKPVLPGEGE 48. peptide 5 of TgR4 MEKEKPVLPGEGEGH 49. peptide 6 of TgR4 KEKPVLPGEGEGHVL 50. peptide 7 of TgR4 KPVLPGEGEGHVLPG 51. peptide 13 of TgR4 HVLPKHETKPALTDEK 52. peptide 14 of TgR4 PKHETKPALTDEKRT 53. peptide 15 of TgR4 HETKPALTDEKRTKP 54. peptide 16 of TgR4 TKPALTDEKRTKPGG 55. Peptide AB_F MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDE KRTKPGGP 56. Peptide A3_B AAGSMEKDKLVLPGE 57. VHH ERB-1G6 EVQLVESGGGLVQAGGSLGLSCAASGRPGRIFTRN SMAWFRQAPGKEREFVASINWSGTSTSYADSVKGR FAISRDNDKNTVYLQMNSLKPEDTAVYYCAADSAL YGSMHKTPADYEYWGQGTQVTVSS 58. VHH ERB-1B11 EVQLVESGGGLVQAGGSLRLTCAASGRTFRRSNM AWFRQPPGKERDFVAAIKWSGSSTNYADSVKGRFT ISRDNDKNTVYLQMNVLKPEDTGVYYCAQESSLYS NYLPVVSSAYDYWGQGTQVTVSS 59. VHH ERB-1A12 EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYFMG WFRQAPGKEREFVAGIIWSGTRTYYVDSVKGRFTIS RDNDKRMVYLQMNSLKPEDTAVYYCAAYKEYYG TPAQLYAAASYDYWGQGTQVTVSS 60. VHH ERB-1F1 EVQLVESGGGLVQAGDSLRLSCAASGRTFSRVTMG WFRQAPGKEREFVAGISWSGTRTDYPDSVKGRFTV SRDNAKKTMWLQMSSLRPEDTAVYHCAADSTLYG SAISNNREAYAYWGQGTQVTVSS 61. VHH ERB-1F2 EVQLVESGGGLVQVGGSLRLSCAASGRTFRRNTIG WFRQAPGKEREFVAAISWSGTRTKYADPVKGRFTI SRDNDKNTAYLQMNTLKPDDTAVYYCAADGALY GSDVSGLARVYDYWGQGTQVTVSS 62. VHH ERB-1H4 EVQLVESGGGLVQAGGSLRLSCVASGRTFSRYTVG WFRQAPGKEREFVAGISWSGSRTSYADSVKGRFTIS RDNDKTTGYLQMNSLKPEDTAVYYCAAITKLYEN NIPRSVSDYALWGQGTQVTVSS 63. VHH ERB-1D7 KVQLVESGGGLVQAGGSLRLSCAASGRTFSRRGM GWFRQAPGKEREFVATIKWSGTSTDYADSVKGRFT ISRDNAKNTVYLQMNNLQPEDTAVYYCAADRQLY RDGYVPLNEYEDWGQGTQVTVSS 64. TgRx M-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-M-E- Xaa8-Xaa9-K-Xaa10-V-Xaa11-P-G-E-G-Xaa12- Xaa13-H-Xaa14-Xaa15-P-K-Xaa16-E-Xaa17-Xaa18- L-T-Xaa19-Xaa20-Xaa21-Xaa22-T-Xaa23-P-Xaa24- Xaa25-P-Xaa26-Xaa27-Xaa28
EXAMPLE 3
[0489] In the present longitudinal study, we imply that detection of BCLA antibodies may conceivably further sensitivity of current tests when combined properly. We have undergone testing of BCLA in the context of mother to child congenital toxoplasmosis. For the moment, only 10 couples per group of mother/child were tested so the results should be considered accordingly. Two groups are compared, one where the congenital toxoplasmosis was confirmed through a persistent Sag1 IgG titer in the child's sera long after birth, the other where congenital toxoplasmosis was excluded when the sera of the child became negative to Sag1 over time (Lebech M et al., 1996).
[0490] As shown by the comparative titration of Toxo IgGs by Vidas® and Architect®, at birth, infants in both groups share comparable titers with no discernable profile (
[0491] This observation implies that the child neo-synthetizes specifically anti-BCLA IgGs prior to birth and indicates that strong BCLA reactivity can further orient the diagnosis of congenital toxoplasmosis at the moment of birth.
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