Patent Application
20140106987
The present invention relates to the field of immunodiagnostic and/or prognostic of liver autoimmune diseases.
Autoimmune Liver Diseases (AILD) are chronic and progressive disorders with a poorly understood etiology. The most common AILD are Autoimmune Hepatitis (AIH) and Primary Biliary Cirrhosis (PBC).
Autoimmune Hepatitis (AIH) is a chronic necro-inflammatory disease and one of the most common autoimmune liver diseases AIH has an incidence of 1-2 per 100,000 per year, and a prevalence of 1-10/100,000. As with most of the other autoimmune diseases, it affects women more often than men (80%), with a sex ratio of about 3:1 (female to male) (Czaja A J. et al., 2010, Gastroenterology; Makol et al., 2011, Hepatitis research and treatment). Histologically it is characterized by: interface hepatitis and plasma cell infiltration; hypergammaglobulinemia is often present; a number of autoantibodies can be detected such as antinuclear antibodies (ANA), anti-Smooth Muscle Antibody (SMA), liver/kidney microsomal antibody (LKM-1), LC1, anti-actin, anti-ASGPR (Bogdanos D P. et al., 2009, Semin Liver Dis). Primary Biliary Cirrhosis (PBC) is a slowly progressing disease causing the destruction of small and medium-size intra-hepatic bile ducts (Selmi C. et al., 2011, Imm Cell Bio). It affects women in 90% of cases. The prevalence is estimated at 0.6-40/100.000. Ursodeoxicholic acid has been shown to improve serum biochemistry, histology and patient's survival (Muratori L. et al., 2010, Dig Liver Dis; Muratori L. et al., 2008, Clin Liver Dis). It is characterized by: anti-mitochondrial antibodies—AMA—(˜90%) and intrahepatic cholestasis (increased alkaline phosphatase—Alk Ph—, normal ultrasonographic—US—scan).
The detection of the AMA autoantibodies is performed routinely by immunofluorescence on fresh multi-organ sections (liver, kidney, stomach) from rodents, but this technique may present many intrinsic problems such as standardization and interpretation of the immuno-morphological patterns (Bogdanos et al., 2008, WJG). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an internationally representative committee to define guidelines and develop procedures and reference standards for more reliable testing (Vergani et al., 2004, Journal of hepatology). In recent years, some AILD target-autoantigens have been identified and characterized (Zachou, K., et al., 2004; J of Autoimm Dis), but little is known on their pathogenetic role, and probably many autoantigens are still unknown. For autoantibodies to have a pathogenetic role, two features have to be met: (i) the autoantigen should be expressed on the target organ and exposed to autoantibodies, (ii) the autoantibodies should have functional activity. Song Q. et al. (2010, J. Proteome Res) described the identification of highly specific biomarkers and their validation for AIH. This study demonstrates that the combination of six autoantigens can be used to diagnose AIH-positive serum samples and that these autoantigens can be effectively used in protein microarray assays, as well as, in traditional ELISA-based assays.
US2009/0023162 discloses the methods for the identification of atypical antineutrophil cytoplasmic antibodies (ANCA), kits suitable for the same and application of said methods to the diagnosis of chronic inflammatory intestinal diseases and autoimmune liver diseases.
RU 2247387 (C1) provides a method involving determination of anti-mitochondrial antibodies, immunoglobulins such as IgA, IgM, IgG, gamma-globulins, anti-gliadin antibodies, and circulating immune complexes for the diagnosis of autoimmune liver injuries in patients with chronic hepatitis.
To date, however, there are no early and precise assays that can be used to identify individuals carrying or at risk of developing AILD. An early diagnosis is clearly important.
Dalekos G. 2002 European J. Int. Medicine, vol. 13, n. 5, pp. 293-303 and Jones D. E. 2000 Journ. Clin. Pathol. Vol. 53, n. 11, pp. 813-821 disclose some autoantigens in AIH and PBC.
Though the identification of some autoantibodies is within prior art documents, there is still the need to identify novel biomarkers, namely autoantibodies, to diagnose the liver autoimmune disease (AILD) and/or to discriminate between autoimmune and other liver pathologies, and/or to monitor the efficacy of patient treatments of liver autoimmune disease and the disease progression. In the present invention a subject with autoimmune liver disease is named “AILD or hepatic autoimmunity patient” and is affected by autoimmune liver disease, including AIH or PBC.
In the present invention, a panel of 17 autoantigens was identified in patients with AILD by protein array. In addition, 6 out of the 17 autoantigens were also validated by Dissociation Enhanced Lanthanide FluoroImmunoAssay method (DELFIA®), in patients diagnosed with liver autoimmune diseases, and showed individual sensitivities ranging from 42% to 74%. The combined assessment of these six autoantigens displays a 82%±4% sensitivity and almost 90%±3% specificity.
These six autoantigens represent novel markers of liver autoimmunity, such as AIH and PBC. These markers can display much higher sensitivity and specificity (Vs other diseases such as HCV and HBV) when compared to the benchmark markers (CYP2D6 & ASGPR, as described in the Methods section). Therefore, the autoantigens identified in the present invention are valuable tools for the development of a new serological assay that is easy to perform and is highly specific for AILD diseases. This assay could significantly contribute to an improvement of AILD diagnosis and to a discrimination between PBC and AIH.
It is therefore an object of the present invention an in vitro method of diagnosis or prognosis or evaluation of risk to develop a liver autoimmune disorder belonging to the group of AIH and PBC in a subject, comprising the steps of:
a) contacting a biological sample from the subject with a protein comprised in the group of: a protein having the amino acid sequence SEQ ID No. 1, an allelic variant, an orthologous, at least one immunological fragment or a functional equivalent thereof, under conditions appropriate for binding of autoantibodies, if present in the biological sample, to said protein, and
b) detecting the presence of bound autoantibodies.
In the context of the instant invention the term “protein” includes:
In a preferred embodiment, step a) is performed by contacting said biological sample with the protein having the amino acid sequences SEQ ID No. 1 and at least one further protein selected from the group of 16 proteins having the amino acid sequences SEQ ID No. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 22, 25, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
In a further embodiment step a) is performed by contacting said biological sample with three proteins having the amino acid sequences SEQ ID No. 1, 11, 17 allelic variants, orthologous, immunological fragments or functional equivalents thereof.
In a further preferred embodiment step a) is performed by contacting said biological sample with four proteins having the amino acid sequences SEQ ID No 1, 10, 11, 17, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
In a further preferred embodiment step a) is performed by contacting said biological sample with six proteins having the amino acid sequences SEQ ID No 1, 6, 8, 10, 11, 17 allelic variants, orthologous, immunological fragments or functional equivalents thereof.
Preferably the biological sample is comprised in the group of blood, serum, plasma, urine, saliva, mucus, or fractions thereof.
Preferably the biological sample is from an adult or from an adolescent.
Preferably the detection of said bound autoantibodies is performed by means of binding to specific ligands. More preferably the ligands are conjugated with detecting means.
It is another object of the invention a method of monitoring an autoimmune liver disorder after treatment with surgery and/or therapy in a subject with said autoimmune liver disorder, comprising the step of following the modulation of antibodies as disclosed.
In a preferred aspect said proteins or immunological fragments or functional equivalents thereof are displayed on one or more protein microarrays.
It is another object of the invention the use of a protein microarray comprising at least the proteins as defined or immunological fragments or functional equivalents thereof for performing the method according to the invention.
It is another object of the invention the use of a solid support for an immunodiagnostic assay comprising at least the proteins as defined or immunological fragments or functional equivalents thereof for performing the method according to the invention.
It is another object of the invention the use of an immunodiagnostic kit comprising the above solid support and detecting means for performing the method according to the invention.
In the present invention a subject with autoimmune liver disease is named “AILD or hepatic autoimmunity patient” and is affected by any autoimmune liver disease, as AIH or PBC.
HCV patients are patients diagnosed with hepatitis C and displaying autoreactive antibodies, or patients with HCV infection or patients with hepatitis C and non hepatic autoimmune diseases such as crioglobulinemia or thyroid dysfunctions.
The invention will be now described by means of non limiting examples referring to the following figures.
Samples used for this study were collected in five different hospitals: i) Policlinico Ospedale Maggiore, Transfusional Unit, Milan; ii) Hepatology Unit, University Hospital, Pisa, Italy; iii) Sant'Orsola-Malpighi University Hospital, Bologna, Italy; iv) Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano, Italy; v) Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), University of Firenze, Italy. For the discovery phase, 218 sera were used (15 AIH, 15 PBC 78 HD, 110 HCV), while for the validation phase 224 sera were used (50 AIH, 50 PBC, 50 HD, 50 HCV, 24 HBV). Table 1 reports the clinical characteristics and ages of the patients and donors enrolled in this invention, for the microarray analysis (Example 2-3).
Genes whose translated products carry a secretion signal peptide or at least one transmembrane domain were selected, cloned and expressed in a high through-put system as histidine-tagged products as described (Grifantini R. et al., 2011, Journal of proteomics). 1626 full-length proteins or protein domains were expressed in E. coli and purified from the bacterial insoluble fraction by Immobilized metal ion affinity chromatography (IMAC, GE).
Human, viral or bacterial proteins were used as biological or technical controls in the microarray. In particular genes encoding for Core protein and Non-structural proteins NS3 (from HCV genotype 1), NS3-4a (from HCV genotype 2) NS5b (from HCV genotype 1), Tetanus toxin and H1N1 were subcloned in E. coli strain DH5α and expressed in BL21(DE3), respectively. Bovine Serum Albumin (BSA), Human Serum Albumin, Human Glutathione-S-Transferase and Protein A from Staphylococcus aureus were purchased from Sigma.
For DELFIA® experiments, Pyruvate DeHydrogenase (PDH) protein was purchased by Sigma, while genes encoding Cytochrome P450 2D6 (CYP2D6) and asialoglycoprotein receptor 1 (ASGR-1) from Ultimate™ Human ORF Clones were purchased by Invitrogen and were subcloned in E. coli strain DH5α and expressed in BL21(DE3), respectively. All the corresponding proteins were purified by affinity chromatography on IMAC resin.
Purified recombinant proteins (10-15 μg total protein), obtained as described above, were stored at 4° C. and analyzed by SDS-PAGE (Criterion PAGE system Bio-Rad) followed by Coomassie Blue staining of the gel immediately before spotting them, to assess their integrity, and purity level. Proteins showing purity levels >70% (ChemiDoc™ XRS, Quantity One® software; Bio-Rad) were used for protein array preparation.
For Western blot analysis, aliquots (0.5 μg) of the proteins were resolved on 4-12% pre-cast SDS-PAGE gradient Tricine gels under reducing conditions, and electroblotted onto nitrocellulose membranes (Bio-Rad), according to the manufacturer's instructions. The membranes were blocked with 5% non-fat milk in 1×PBS plus 0.1% Tween 20 (TPBS) for 1 h at room temperature, incubated with the α-His mAb (GE-Healthcare) diluted 1:1000 in 3% non-fat milk in TPBS 0.1% for 1 h at room temperature, and washed three times in TPBS 0.1% (
Protein MicroArrays were generated by spotting the 1626 affinity-purified recombinant proteins (0.5 mg/ml, in 6M Urea) in 4 replicates on nitrocellulose-coated slides (FAST slides, GE-Healthcare) using Stealth SMP3® spotting pins (TeleChem International, Sunnyvale, Calif.) and a Microgrid II microarray contact printer (Biorobotics), resulting in spots of approximately 130 μm in diameter. As experimental positive control, to assess the sensitivity and reproducibility of the arrays and for signal normalization, a curve of human IgG(s) at 11 different concentrations (solutions from 0.001 to 1 mg/ml) was spotted on the arrays in 8 replicates (in 6M Urea) and detected with Alexa-647 conjugated α-Human IgG secondary antibody (Invitrogen). As negative controls the spotting buffer alone, was printed and used to assess possible non-specific signals due to cross contamination.
A quality control of the spotting procedure was performed on 10% of randomly chosen slides, by confirming the presence of the total immobilized proteins using the α-His mAb, followed by detection using a Alexa-647 conjugated α-Human IgG secondary antibody (
Incubation was automatically performed with a TECAN Hybridization Station (HS4800™ Pro; TECAN, Salzburg, Austria). The microarray slides were prewashed 3 min in TPBS 0.1% Tween 20, and saturated with BlockIt™ Microarray Blocking Buffer (Arrayit Corporation) for 45 min at 25° C. under mild agitation. After injection of 105 μl of individual serum (diluted 1:300 in Blocking Buffer plus 0.1% Tween 20), incubation was performed at 25° C. for 45 min with low agitation. The microarrays were washed at 25° C. in TPBS for three cycles of 1 min wash time and 30 sec soak time.
Afterwards the microarray slides were incubated at 25° C. for 1 with Alexa-647 conjugated α-human IgG (Invitrogen) diluted at 1:800 in Blocking Buffer in the dark. The microarrays were again washed at 25° C. in TPBS for two cycles of 1 min wash time and 30 sec soak time, in PBS for two cycles of 1 min: 30 sec and finally in milliQ sterile water for one cycle of 15 sec.
The microarray slides were finally dried at 30° C. under nitrogen for 2 min, and scanned using a ScanArray Gx PLUS (PerkinElmer, Bridgeport Avenue Shelton, USA). 16-bit images were generated with ScanArray™ software at 10 μm per pixel resolution and processed using ImaGene 8.0 software (Biodiscovery Inc, CA, USA). Laser of 635 nm was used to excite Alexa-647 dye. The fluorescence intensity of each spot was measured, signal-to-local-background ratios were calculated by ImaGene, and spot morphology and deviation from the expected spot position were considered using the default ImaGene settings.
For each sample, the Mean Fluorescence Intensity (MFI) of replicated spots was determined, after subtraction of the background value for each spot, and subsequently normalized on the basis of the human IgG curve to allow comparison of data from different set of experiments (Bombaci M. et al., 2009, PLoS One). Briefly, the MFIs values of IgG, spotted at different concentrations, were best fitted by a sigmoid curve, using a maximum likelihood estimator (Harris J W et al., 1998, Handbook of Mathematics and Computational Science). The experimental average IgG curve of each slide was adjusted on the reference sigmoid IgG curve, and the background-subtracted MFI values of each protein were normalized accordingly. On the basis of these results, a normalized MFI value of 4.000 was chosen as the lowest signal threshold for scoring a protein as positively recognized by human sera. For each protein, a Coefficient of Variation (CV %), was calculated on the four replicate spots, for intra-assay reproducibility (Bombaci et al., 2009, PLoS One).
Recognition Frequency was defined as the percentage of sera reacting with a particular antigen in protein array with a MFI 4.000, and it was calculated for each group of sera. TIGR Multiexperiment Viewer (version MeV4.5) software (Saeed, A. I., et al., 2006, Methods in enzymology) was used to perform an unsupervised bi-dimensional hierarchical clustering.
The DELFIA® assay is a time-resolved fluorescence method that can be used to study antibody binding to solid-phase proteins or peptides. The purified recombinant proteins were used at a concentration of 20 μg per milliliter (Frulloni L. et al., 2009, N Engl J Med) in 6 M urea to coat DELFIA® plates (PerkinElmer). Plates were then blocked for 1 hour at 37° C. with a blocking reagent (PerkinElmer). The blocking buffer was than discarded, and the serum samples were diluted in a 1:300 solution in phosphate buffered saline plus 1% bovine serum albumin (Sigma), plus 0.1% Tween 20 (Sigma) and incubated on the plates 1 hour at 37° C. Plates were then washed 5 times with washing buffer (PerkinElmer). Bound antibodies were detected with europium-labeled α-human IgG serum (1:500 in diluting buffer, PerkinElmer), incubated 30 min at RT in the dark. The wells were again washed in the same washing buffer. After a 10 min incubation at RT, the plates were read on a Infinite F200 PRO instrument (Tecan). Fluorescence intensity values higher than the mean of buffer plus 3 standard deviations were considered to be positive.
Results of Protein Microarray and DELFIA® experiments from sera of patients and healthy donors were compared using the two-tailed χ2 test, the Student's t-test or the Fisher's exact tests. The ANOVA test was used for all the others analyses. The Benjamini-Hochberg correction for multiple testing was used for the analysis of microarray data. Statistical analysis was carried out with the use of GraphPad Prism 5 software, version 5.01. To evaluate the performance of autoantigens combinations in discriminating AILD patients from Healthy donors or HCV patients, logistic regression analysis was performed with R. We the signals of respectively 6, 4 or 3 selected autoantigens we created logistic regression models. The probabilities were calculated as follows: p=exp((Σ(bixi)+c)/(1+Σ(bixi)+c), where p is the probability of each case, i=1 to n; b is the regression coefficient of a given autoantigen, x is signal intensity and c is a constant generated by the model. ROCR package was used to obtain the ROC curves of the models and the Area Under Curve (AUC) values (Sing T. et al. 2005, Bioinformatics).
To study the serological profile of patients diagnosed with autoimmune liver diseases versus a panel of self proteins, the authors developed a protein array by printing 1626 human recombinant (see details in Materials and Methods section) products that corresponded to 1371 distinct human proteins distributed as shown in Table 2.
Briefly, 1329 of the 1371 the proteins were first selected through a bioinformatic analysis of the whole human genome as translated sequences carrying i) signal peptides, ii) at least one transmembrane domain, iii) having unknown biological function. Fortytwo of the 1371 proteins had a well known immunological function, CD number assigned and were all surface exposed. The majority of printed human proteins were expressed as N-terminal His-tag fusions while 48 proteins where expressed as double tagged fusions, with an N-terminal glutathione S-transferase (GST) and with C-terminal Histidines. Proteins obtained after affinity purification from the bacterial insoluble fraction showed purity levels >70%, as estimated by densitometric scan of SDS-PAGE gels (see Materials and Methods) (
Protein arrays were prepared by printing onto nitrocellulose-covered glass slides four replicates of each protein. In addition, we included in the array, several biological and technical controls including human, viral and bacterial proteins (Materials and Methods). Replicates were randomly distributed to get optimal signal reproducibility. Moreover. eleven different amounts of human IgGs at a known concentration (from 8.24×10−4 to 7×10−1 ng of immobilized protein/spot) were printed also on the array in eight replicates (
The quality of the immobilized proteins on the arrays were determined by probing 10% of the slides with an anti-His mAb, and 89% of the proteins produced signals that were significantly above the background (
In an attempt to determine a panel of autoantigens differentially recognized by patients sera with AILD compared to healthy individuals (HD), the protein microarrays were probed with a sample set (defined as Training set as indicated in Table 1 in Materials and Methods) comprising 15 sera patients with AILD, and 39 sera from healthy donors. The clinical characteristics of each group of sera are summarized in Table 1.
Sera reactivity was evaluated by detecting total IgG bound to each protein spot using Alexa-647 conjugated α-human IgG and measuring the fluorescence intensity (FI) values for each protein. To compare data from different experiments, we used a normalization method, as previously described (Bombaci M et al., 2009, PLoS One). Briefly, the experimental average IgG curve of each slide was adjusted on a reference sigmoid IgG curve, and the background-subtracted mean fluorescence intensities (MFIs) values of each protein were normalized accordingly. On the basis of these results, a normalized-MFI value of 4000 was chosen as the lowest signal threshold for scoring a protein as positively recognized by human sera.
First of all, total reactivity of patients against healthy donors sera was evaluated. Representative images of a zoomed grid of the microarray probed with sera derived from healthy donors and AILD patients are shown in
Significant divergences between patients and healthy donors were also observed in terms of the recognition frequencies, indicating differential protein-specific IgG levels
Having previously established that patients with AILD of Training set displayed an increased autoreactivity when compared to healthy donors, the authors selected the autoantigens specifically recognized by those patients. Following normalization, individual autoantigens from protein microarray were ranked according to (i) the recognition frequency and (ii) the mean fluorescence intensity of AILD patients as compared to the healthy donors.
To be considered of potential interest, the antigen-specific responses had to occur with a significantly higher signal intensity in patients sera than in healthy donors sera (T test's p val <0.01). In particular, the proteins should be recognized in less than 10% of the healthy donors sera and in at least 25% of sera from patients groups (Fisher test's pval <0.01). By using this approach, the authors identified 25 distinct proteins (Table 3) showing a higher immunoreactivity with AILD patients .compared to controls sera.
In order to confirm the above results, a different set of proteins spotted in the microarray was used. This microarray now included all proteins identified as more reactive in the training set including the 24 proteins as indicated above (Table 4).
In order to confirm the above results, the authors probed the focused protein microarray with a second serum sample set (Test Set as indicated in Table 1 Material and Methods) comprising other 15 sera of patients with AILD and 39 serum samples from healthy subjects. Following the same normalization and using criteria described above (see Materials and Methods), the authors confirmed that the 25 autoantigens were differentially recognized by patients compared to healthy donors with statistical significance. Interestingly, in unsupervised hierarchical clustering analysis these autoantigens allowed for good discrimination of the two populations of sera in both sample sets, as shown in
Having identified 25 autoantigens highly recognized by AILD patients, the authors asked whether non-autoimmune liver disease patients had an overlapped recognition pattern. They therefore tested the same microarray with sera from 110 patients with chronic HCV infection (Table 1).
After having identified a total of 17 auto-antigens specific for autoimmune patients by protein microarray, the authors validated the results obtained by using the Dissociation-Enhanced Lanthanide Fluorescence ImmunoAssay method (DELFIA®) assay method (Materials and Methods).
By this assay the authors screened an independent sample set (Validation set as indicated in Table 1, Materials and Methods) comprising 100 AILD patients (50 AIH and 50 PBC), 50 healthy donors and 74 patients with chronic viral hepatitis (50 HCV and 24 HBV) measured by time-resolved fluorescence. All sera were tested at a dilution of 1:300 as described in Materials and Methods, and the antigen-specific IgG responses to each of the 17 selected autoantigens was measured by time-resolved fluorescence. Reproducibility of the results was confirmed using duplicate sample of selected sera.
All 17 antigens displayed higher mean fluorescence intensity compared to HD and chronic viral hepatitis (
AAntigens used for combination assays comprising 6 (x), 4 (∘) and 3 (•) markers respectively.
These top 6 antigens, showed high sensitivity (from 44 to 74% of positive AILD patients) and specificity (from 92 to 100% of negative HD). Interestingly, individual sensitivity was comparable to that obtained in our hands by 3 known AILD markers, CYP2D6 (Cytochrome Peroxidase 2D6), AGPR-1 (Asialo-Glycoprotein Receptor 1) and PDH (Pyruvate DeHydrogenase), (Jensen, D M, 1978, The New England journal of medicine; Van de Water J. et al., 1993, The Journal of clinical investigation), while individual specificity was far better for our candidates compared to the benchmarks.
The authors next assessed the discrimination power of combinations of the six autoantigens. We therefore tested combinations of three (YM0078, YM1672, YM2046), four (YM0078, YM1652, YM1672, YM2046) or six proteins (YM1672, YM0078, YM2046, YM1652, YM1503, YM1602).
| Number | Date | Country | Kind |
|---|---|---|---|
| 11170252.8 | Jun 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/061313 | 6/14/2012 | WO | 00 | 12/10/2013 |
