WO2011124652A1 - Peptide immunodominant du virus du polyôme jc et son utilisation - Google Patents

Peptide immunodominant du virus du polyôme jc et son utilisation Download PDF

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WO2011124652A1
WO2011124652A1 PCT/EP2011/055448 EP2011055448W WO2011124652A1 WO 2011124652 A1 WO2011124652 A1 WO 2011124652A1 EP 2011055448 W EP2011055448 W EP 2011055448W WO 2011124652 A1 WO2011124652 A1 WO 2011124652A1
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seq
jcv
pml
peptide
vpl
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PCT/EP2011/055448
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Paola Maria Piera Cinque
Manuela Testa
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Fondazione Centro San Raffaele Del Monte Tabor
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to an immunodominant peptide of polyomavirus and its use in an assay for the assessment of the JCV-specific T cell responses.
  • such assay can be used for predicting the risk of developing or for monitoring progressive multifocal leukoencephalopathy as well as other pathologies involving the JC virus.
  • PML Progressive multifocal leukoencephalopathy
  • CNS central nervous system
  • JC virus JC
  • JCV JCV encephalopathy
  • PML usually develops in the context of immune disorders, such as HIV infection, hematological malignancies or transplantation (Richardson 1983; Garcia-Suarez 2005; Cinque 2009).
  • Interest in PML has increased in recent years because of several cases developing in patients receiving immunomodulant treatments with monoclonal antibodies, such as the alpha-4 integrin antagonist natalizumab, used in multiple sclerosis (Kappos 2007; Hartung 2009), the CD 11a antagonist efalizumab, used for treatment of psoriasis (Molloy 2009) and, possibly, the anti-B-cell antigen CD20 rituximab rituximab, used in the treatment of lymphoroliferative and autoimmune disorders (Carson 2009).
  • monoclonal antibodies such as the alpha-4 integrin antagonist natalizumab, used in multiple sclerosis (Kappos 2007; Hartung 2009), the CD 11a antagonist efalizumab, used for treatment of psorias
  • PML is in most of the cases progressively fatal within a few months from onset.
  • JCV-specific T-cell immunity has so far been assessed in PML using several approaches, including T-cell lymphoproliferation (studying the CD4 cell responses, Gasnault 2003), cytotoxic T cells (CTL) chromium release or tetramer assays (measuring the CD8 cell activity, Du Pasquier 2004) and the functional cytokine secretion and Elispot assays, measuring both CD4 and CD8 responses (Khanna 2009).
  • CTL cytotoxic T cells
  • tetramer assays measuring the CD8 cell activity, Du Pasquier 2004
  • Crealnik et al. have identified two main A*02-restricted JCV 9-aa epitopes of the JCV capsid protein VPl, corresponding to the VPl-p36 and VPl-pl00 peptides.
  • measuring the T-cell response through an easy and rapid procedure would represent an additional objective and non invasive approach that enables real-time monitoring of PML patients and help provide prognostic information in real-time.
  • most of the PML patients show low JCV-specific CTL responses at the time of symptoms onset, thus measuring these responses could also be useful to predict patients at risk for PML.
  • treatments associated with PML onset such as natalizumab or rituximab, both before starting and during treatment (Chen 2009). Indeed, criteria for risk stratification have been proposed for patients starting natalizumab treatment or already being treated with this drug.
  • Presence of JCV- VP 1 IgG and previous treatment with immunosuppressive drugs are associated with a greater risk to develop PML.
  • the risk seems to increase over time, with most of the cases of PML occurring after 24 months of natalizumab therapy. Therefore, the risk appears to be higher in natalizumab treated patients with positive anti-JCV IgG and who have previously been treated with immunosuppressive drugs (Gorelik 2010).
  • the study of T-cell responses during the course of JCV infection from chronic persistent infection to widespread CNS active infection, would be key to understand the host- pathogen relationships and better define the immunological background for PML to occur.
  • Elispot assays measuring both CD4 and CD8 responses. Recently, Elispot has been used to measure IFN-gamma release by JCV-specific T-cells after PBMC stimulation with overlapping peptide pools from both VP1 and Large T-antigen (Chen 2009; Khanna 2009; Jilek 2010). Elispot offers a relatively easy, fast and quantitative measure of the immune responses against specific antigens.
  • the international application WO2010/090757 describes methods and compositions for determining whether a subject is at risk for PML, including subjects being treated with immunosuppressants by determining whether the subject harbors a JCV variant with reduced binding for sialic acid relative to a normal JCV.
  • WO2010/100182 describes an immunological method for detecting active JCV infection by screening for the presence of activated T lymphocytes against JC virus in a blood sample of a patient, by exposing the blood sample to epitopes of the virus for up to 48 hours.
  • WO2009/105212 discloses methods and kits for testing for the presence or absence of a polyomavirus, such as BKV, in a sample.
  • the methods and kits are useful for quantifying BKV and differentiating BKV from JCV.
  • WO2009/038684 relates to compositions, methods, and kits for treating subjects infected by or at risk of infection with a DNA virus (e.g., a JC Virus or a BK virus). Aspects of the invention are useful to prevent or treat DNA virus associated conditions (e.g., PML) in subjects that are immunocompromised. Compositions are provided that inhibit intracellular replication of DNA viruses.
  • a DNA virus e.g., JC Virus or a BK virus.
  • a DNA virus associated conditions e.g., PML
  • Compositions are provided that inhibit intracellular replication of DNA viruses.
  • WO1992/019774 methods for detecting the propensity for an individual to be affected by a polyomavirus are disclosed.
  • the methods include an assay wherein a biological specimen from a female is contacted with at least one probe capable of determining whether the female has been exposed to a polyomavirus.
  • a method for prophylactically treating the female is also described.
  • US2009/0099335 relates to HLA-A*02-restricted cellular epitopes within the VP1 polypeptide of human polyomaviruses, which are useful as diagnostic reagents for virus infection.
  • Preferred peptides correspond to amino acids residues 107-116, 108-116 and 44- 52 of BKV VP1, and are processed in vivo in natural infection with BKV.
  • Effector T cell populations stimulated by the peptides represent functional CTLs as assessed by cytotoxicity and cytokine production, and are reactive against cells presenting both the BKV peptides above and the JC virus homo log sequences.
  • JCV-VP1 specific responses consisted in the screening of a library of seventy 10-mer peptides spanning the entire coding sequence of the major viral capside protein- 1 (VP1, total length 354 aa), in search for immunodominant peptides.
  • the present invention relates to an assay for assessing the JCV-specific T-cell responses and identifies immunogenic JCV-specific VP1 epitopes.
  • VP1 major viral capside protein- 1
  • PBMC peripheral blood mononuclear cells
  • JCV strains present in the cerebrospinal fluid (CSF) of PML patients carry one of several VP 1 -specific substitutions at critical sites for sialic acid binding, with S269F mutation being the most commonly observed and, together with mutations at positions 265 and 267 (N265D, S267F/L/Y) accounting for 42% of all observed VP1 mutations (Gorelik, 2011).
  • the VPl-p261 peptide of the invention is located in the proximity of the VP1 binding pocket with the sialic acid cell receptor and includes aa at position 265, 267 and 269 in its core region.
  • VP1- p261 a novel VP1 immunodominant peptide, named VP1- p261, which is of relevance because:
  • GMFTNRSGSQ (SEQ ID No.12), GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24) for use in the diagnostic and/or prognostic of the following pathologies: progressive multifocal leukoencephalopathy (PML), cerebellar granule cell neuronopathy and/or JCV nephropathy.
  • PML progressive multifocal leukoencephalopathy
  • JCV nephropathy JCV nephropathy
  • PML progressive multifocal leukoencephalopathy
  • cerebellar granule cell neuronopathy and JCV nephropathy share common features in that they are all mediated by JCV and result in JCV reactivation.
  • the peptide consists of the sequence selected from the group of: GMFTNRSGSQ (SEQ ID No.12), GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24).
  • the peptide consists of the sequence GMFTNRSGSQ (SEQ ID No.12).
  • the diagnostic and/or prognostic of said pathologies is performed by measuring JCV T cell response.
  • the JCV T cell response is measured by means of an Elispot assay.
  • the Elispot assay is an interferon-gamma Elispot assay.
  • JCV T cell response may also be measured by methods known by the skilled person in the art such as intracellular staining methods, cytofluorimetric methods, measurement of lymphocytes proliferation.
  • GMFTNRSGSQ (SEQ ID No.12), GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24) for predicting the risk of developing progressive multifocal leukoencephalopathy (PML) in a subject being anti-JCV IgG positive.
  • PML progressive multifocal leukoencephalopathy
  • the peptide consists of the sequence selected from the group of: GMFTNRSGSQ (SEQ ID No.12), GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24).
  • the peptide consists of the sequence GMFTNRSGSQ (SEQ ID No.12).
  • the subject has been previously exposed to an immunomodulant treatment.
  • the immunomodulant treatment is natalizumab.
  • the prediction of risk of developing progressive multifocal leukoencephalopathy (PML) in a subject being anti-JCV IgG positive is performed by measuring JCV T cell response.
  • JCV T cell response is measured by means of an Elispot assay.
  • the Elispot assay is an interferon-gamma Elispot assay.
  • It is a further object of the invention a method for monitoring a JCV T cell response in a sample obtained from a subject comprising the steps of:
  • It is a further object of the invention a method for predicting the risk of developing PML in subject being anti-JCV IgG positive or to predict the outcome of PML and/or to monitor the course of PML in a subject affect by PML comprising the steps of: a) isolating a sample of PBMCs or mononucleic cells from the subject;
  • step b) comprises incubating the isolated PBMCs or mononucleic cells with the peptide comprising the sequence GMFTNRSGSQ (SEQ ID No.12) and at least one further peptide comprising a sequence selected from GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24).
  • GMFTNRSGSQ SEQ ID No.12
  • step b) comprises incubating the isolated PBMCs or mononucleic cells with the peptide comprising the sequence GMFTNRSGSQ (SEQ ID No.12) and at least one further peptide comprising a sequence selected from GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGS
  • the peptide comprising the sequence GMFTNRSGSQ (SEQ ID No.12) consists of SEQ ID No. 12.
  • the peptide comprising a sequence selected from GMFTDRSGSQ (SEQ ID No. 20), GMFTNRLGSQ (SEQ ID No. 21), GMFTNRFGSQ (SEQ ID No. 22), GMFTNRYGSQ (SEQ ID No. 23), or GMFTNRSGFQ (SEQ ID No. 24) is a peptide consisting of SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 or SEQ ID No. 24.
  • the mononucleic cells are isolated from cerebrospinal fluid.
  • the JCV T cell response is measured by means of an Elispot assay.
  • the Elispot assay is an interferon-gamma Elispot assay.
  • the methods may comprise obtaining PBMCs from fresh blood or utilizing cryopreserved PBMCs, similarly mononucleic cells may be obtained freshly or may be used after cryopreservation.
  • PBMCs or mononucleic cells may be then incubated with the peptide as defined above on microwell plates coated with anti human interferon- ⁇ (IFNy) antibody, subsequently there may be provided the addition of biotinylated detection-antibody against human IFNy, of streptavidin-AP conjugate and of the substrate to develop the reaction.
  • the method may include counting the spot-forming units (SFU) by an ELISpot reader. Values may be expressed as the mean of duplicate experiments.
  • SFU spot-forming units
  • JCV T cell response may also be measured by methods known by the skilled person in the art such as intracellular staining methods, cytofluorimetric methods, measurement of lymphocytes proliferation.
  • JCV T cell response obtained in PBMCs or mononucleic cells may be analysed at regular intervals (either weekly, or every 2 nd , 3 rd or 4 th week or at longer and variable intervals) during the course of a disease such as PML or during the course of a treatment.
  • This approach is of particular significance when immunesuppression is reduced by pharmacological or other interventions, i.e., initiation of HAA T in HIV-related PML, or, in other patients, interruption or reduction of immunosuppressive treatments, or plasma exchange to eliminate circulating immunomodulant molecules.
  • the analysis of the responses at the different time points may be compared to first and previous examinations to identify relative changes in responses.
  • the appropriate control response may be a response before the start of a treatment, the response in a non JCV IgG positive subject, the response in a healthy subject, a response at various time points during the course of a disease or a treatment.
  • JCV T cell response may be assesses by counting SFU in specific contexts (e.g., treatment with immunomodulant or immunosuppressive drugs) and analysis of subject PBMCs or mononucleic cell may be performed before the initiation of treatment or during treatment. Depending on the results, one could then consider to treat only subjects who show responses against the peptide or the combination of peptides (peptide p261 and its mutated froms). Based on previous experience with other Elispot assays and on the responses observed against VPl-p261 in the JCV IgG negative subject, a value of about 25 SFU x 10 A 6 cells may be chosen as a cut-off to distinguish responses from non-responses. Also, depending on the results, one could consider to withdraw a pharmacological treatment in a subject who shows a significant decrease in the response against the peptide above indicated, e.g., below the cut-off value, during the treatment itself.
  • the present invention refers to definitions as indicated in Cinque 2003.
  • a PML survivor is a patient in whom PML remitted months to years before current evaluation.
  • a PML progressor is a patient in whom PML progressed until to death.
  • the term "Good outcome” means a patient in whom PML would remit (clinical stabilization or improvement), the term “bad outcome” means a patient in whom PML would progress (clinical progression and death).
  • a patient with active PML is a patient with signs of active disease (often at the beginning of the disease and/or close to time of diagnosis) in whom the outcome is still unpredictable, "good” and “bad outcome” are here used to classify patients with active disease at the time of evaluation according to subsequent good or bad disease evolution.
  • the peptide comprising or consisting of SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 21 , SEQ ID No. 22, SEQ ID No. 23 or SEQ ID No. 24 may be used at concentration in a range of 2-4 ⁇ g/mL (per well), preferably at 2 ⁇ g/mL.
  • the incubation time may range between 6 and 36 hours. Longer incubation times of 7 to 10 days can be used if T cell need be expanded in vitro .
  • the number of cells per well may be between 100,000 to 500,000 cells. Preferably 200,000 cells per well are used.
  • Figure 1 Configuration of the 17 10-mer peptide pools created by a matrix method and identified by letters A to S. The number in the table is the number code of each peptide.
  • Figure 2. IFN-gamma Elispot T-cell responses in 7 JCV-antibody positive healthy donors and 9 HIV-positive "PML survivors" to the 17 10-mer peptide pools created by a matrix method and identified by letters A to S.
  • Figure 3 IFN-gamma T-cell responses in healthy donors and PML patients against VP1- p261 peptide.
  • Ab neg HD JCV-VP1 IgG negative healthy donors; Ab neg HIV+ pt: JCV- VP1 IgG negative HIV positive control patients;
  • Ab pos HD JCV-VP1 IgG positive healthy donors;
  • Ab pos HIV+ pt JCV-VP1 IgG positive HIV positive control patients;
  • PML survivors Patients in whom PML remitted months to years before current evaluation; Active PML: Patients with signs of active disease (analysis performed at the beginning of the disease and/or close to time of diagnosis) in whom the outcome is still unpredictable.
  • Prolmmune Class I REVEAL rate assay on and off rates of VPl-p261 binding to HLA-A*02 molecule.
  • A on-rate of VPl-p261 binding
  • B on-rate of intermediate positive control (known T-cell epitope) binding
  • C on-rate of positive control (known T-cell epitope) binding
  • D off-rate of VPl-p261 binding
  • E off-rate of intermediate control binding
  • F off-rate of positive control binding.
  • Figure 6. Elispot responses in CD4-depleted and CD8-depleted blood cell populations.
  • Figure 7. IFN-gamma T-cell responses in HLA-A*02 healthy donors and HLA-A*02 PML patients against the two previously described HLA-A*02 restricted peptides VPl-p36 and VP 1 -p 100 and the novel peptide VP 1 -p261.
  • Figure 8. IFN-gamma T-cell responses against VPl-p261 in patients with active PML at the time of diagnosis according to subsequent disease evolution. Good outcome: patients in whom PML would remit; bad outcome: patients in whom PML would progress.
  • FIG. 9 IFN-gamma T-cell responses against VPl-p261 in sequential samples collected from PML patients at subsequent times following diagnosis
  • Panels A, B, C show the responses in three patients with favorable outcome following initiation of highly active antiretroviral therapy (HAART) in a patient with HIV-related PML (A), following plasma exchange (Plex) in a patient with natalizumab-related PML (B) or following interruption of immunosuppressive (IS) drugs (C); panels D, E, F show the responses in three patients with progressively fatal disease, with non-Hodgkin lymphoma (NHL)(D), or HIV-related PML, despite initiation (E) or continuation (F) of HAART.
  • NHL non-Hodgkin lymphoma
  • E non-Hodgkin lymphoma
  • PBMC peripheral blood cells
  • PBMC peripheral blood cells
  • FBS fetal bovine serum
  • SIGMA fetal bovine serum
  • PenStrep SIGMA
  • Cerebrospinal Fluid was collected from PML patients by lumbar puncture as part of the diagnostic work-up, Patients gave their written informed consent to store CSF aliquots and to analyse CSF for the purposes of the present study Peptides
  • GenBank: AAA82101.1; Frisque 1984 were initially synthesized, to be pooled and analysed according to the matrix method. Subsequently, additional 7-mer, 8-mer, 9-mer and 10-mer peptides, including "mutated" peptides were also synthesized (Primm srl, Milan, Italy). All the peptides had been purified by HPLC, the peptide purity >70%, and were further analyzed by mass spectrometry. The lyophilized peptides were solved in 100% DMSO, tipically 16.5 mg/ml depending on solubility, further diluted with PBS to a final concentration of 4 mg/ml and frozen as aliquots at -20°C. Peptides were pooled (as indicated in Fig. 1) and also tested individually (those indicated in Table 4), at a final concentration of 2 ug/ml.
  • 96-well PVDF Multiscreen-HA plates (Millipore, Italy) were coated with 100 ul of anti- human interferon- ⁇ (IFNy) antibody (5 ug/ml; clone 2G1; Pierce Biotechnology, Rockford, USA) in PBS. The plate was incubated overnight at 4°C. The content of the plate was thoroughly washed with 200 ul/well of PBS to remove unbound antibody, and this procedure was repeated for a total of three washes. After washing, the plate was turned upside down and blotted onto paper towels. The coated wells were blocked with 200 ul media (RPMI-1640 supplemented with 20% FBS, 2 mM L-glutamine and 10% PenStrep).
  • IFNy interferon- ⁇
  • the peptide concentration of 2 ⁇ g/mL was used as the standard, however, it may be used in the range of 2-4 ⁇ g/mL (per well).
  • the incubation time may range from 6 to 36 hours. Longer incubation times can also be used (7 to 10 days) if T cell need be expanded in vitro.
  • the number of cells per well is typically 200,000 cells per well, however the number of cells may range may from 100,000 to 500,000 cells Human Leukocyte Antigen Typing
  • the authors characterized all the donors to verify the specificity of HLA class I binding peptides and to evaluate the strength of the peptide/HLA complex.
  • the authors determined the major histocompatibility complex class I allele A using Sequence Specific Primer (SSP) PCR with HLA EZ-TYPE low resolution kit (GTI Diagnostic ITALIA srl). DNA was extracted from fresh or frozen blood with QIAamp DNA Blood Mini Kit, QIAGEN. Results were analyzed by using the interpretative software EZ-Type.
  • SSP Sequence Specific Primer
  • HLA EZ-TYPE low resolution kit GTI Diagnostic ITALIA srl
  • DNA was extracted from 200 of CSF using the QIAamp Blood Kit (Qiagen) and eluted in a final volume of 50 ⁇ ⁇ .
  • VPl was amplified either using primers flanking the whole VPl gene (full VPl PCR), or, when amplification with this method was not successful, by a semi-nested PCR assay that amplified separately shorter VPl regions (short fragment VPl PCR).
  • the full VPl PCR consisted of a nested assay that used the outer primers VPl- LF and VP1-LR, amplifying a 2027 bp fragment; and the inner primers VP1-SF and VP1- SR, amplifying a 1233 bp long fragment (Table 2).
  • the PCR reaction mixture consisted of 5 ⁇ . of 10X PCR buffer, 4 mM of each dNTP, 0.7 ⁇ of primers VP1-LF and VP1-LR in the first round and primers VP1-SF and VP1-SR in the second round, 1.25 unit of Platinum Taq HF (Invitrogen) and 1 of extracted DNA (first round) or 2.5 ⁇ of amplified product (second round) in a total volume of 50 ⁇ ,. Cycling parametres were (for both first and second round) 30 cycles at 94 °C for 20 sec, at 58 °C for 30 sec and at 68 °C for 90 sec in an automated thermal cycler (Applied Biosystems).
  • the short fragment PCR consisted of a semi-nested PCR that used primers VPl-1 and VPl-4a in the first round, amplifying a 797 bp fragment, followed by two semi- nested assays with primers VPl-1 and VPl-2a, amplifying a 481 bp-long fragment, or with primers VPl-1.5 and VPl-4a, amplifying a 490 bp-long fragment (Zheng 2005, Table 2).
  • Table 2 Sequences of primers used for amplifiction of JCV-VP1 from cerebrospinal fluid of PML patients.
  • the PCR reaction mixture consisted of 2.5 ⁇ 10X PCR buffer, 200 ⁇ of each dNTP, 1.5 mM MgC12, 0.5 ⁇ primers and 1.25 unit of AmpliTaq Gold DNA Polymerase (Applied Biosystems).
  • 4 ⁇ of extracted DNA were added in a total volume of 25 ⁇ , and the cycling parameters were 20 cycles at 94 °C for 20 sec, at 55 °C for 30 sec and at 68 °C for 90 sec in an automated thermal cycler (Applied Biosystems).
  • This first step PCR product was purified with ExoSAP-IT PCR Clean-up Kit, the protocol consists of a single pipetting step (enzyme mixture addition), a 30-min incubation at 37 °C followed by enzyme inactivation at 80 °C for a further 15 min.
  • the second round of semi-nested PCR 4 ⁇ of cleaned PCR product were added in a total volume of 25 ⁇ , and the cycling parameters were 40 cycles at 94 °C for 20 sec, at 62 °C for 30 sec and at 68 °C for 90 sec in an automated thermal cycler (Applied Biosystems).
  • Mini-prep DNA was prepared (Qiagen) from colonies containing the cloned VP1 PCR product. Two to 48 clones were sequenced for each sample sample (median 23). Following translation of the VP1 sequences, amino acid mutations were marked by comparison to the large selction of VP1 sequences from PML and non-PML cases (Sunyaev 2009). Only mutations present in more than one clone for sample were considered.
  • VP1 major viral capside protein- 1
  • PBMC peripheral blood mononuclear cells
  • VPl-p23 The authors found a 100% identity for the VPl-p231, but of 70% (7/10 aa homology between the two viruses) for VPl-p261.
  • sequence homology of VPl-p36 was of 89%> (8/9 aa homology) and of 78% for VPl-pl00 (7/9 aa homology).
  • 10-mer peptides like those included in the JCV-VP1 peptide library, are more likely to be recognized by MHC Class I antigens and thus mediate CD8+ T cell responses.
  • MHC Class II antigen mediated CD4+ T cell response may also be possible, depending on the features of the peptide. Indeed, the authors believe that the response against the authors' peptide VPl-p261 is predominantly mediated by MHC class II antigens for the following reasons:
  • JCV mutant virus containing aminoacid substitutions at several specific JCV VPl positions including, among the others, aminoacids 55, 60, 267 and 269, are positively selected during PML from pre-existent wild-type virus within the patient (Sunyaev 2009, Gorelik, in press). Notably, these amino acids are located within the outer JCV VPl loops at critical sites for cell binding with the sialic acid residues of cell receptors (Gee 2004).
  • Table 4 IFN-gamma T-cell responses in PML patients with known PML-associated CSF JCV-VP1 mutations.
  • Active PML-Progr. The analysis was performed in patients with signs of active disease (at the beginning of the disease and/or close to time of diagnosis), who would later progress (bad outcome), Active PML-Survivor: The analysis was performed in patients with signs of active disease (at the beginning of the disease and/or close to time of diagnosis), who would later remit (good outcome); PML-Survivor: patients in whom PML remitted months to years before current analysis.
  • Elispot assays measuring both CD4 and CD8 responses. Recently, Elispot has been used to measure IFN-gamma release by JCV-specific T-cells after PBMC stimulation with overlapping peptide pools from both VP1 and Large T-antigen (Chen 2009; Khanna 2009; Jilek 2010). Elispot offers a relatively easy, fast and quantitative measure of the immune responses against specific antigens.
  • JCV- VP 1 specific responses consisted in the screening of a library of seventy 10-mer peptides spanning the entire coding sequence of the major viral capside protein- 1 (VP1, total length 354 aa), in search for immunodominant peptides.
  • VPl-p231 Another peptide (VPl-p231) was also identified through the matrix method of the present inevntion, and found to elicit T-cell responses in most of the subjects examined. However, the sequence of VP 1-231 was identical to the homo log sequence of BKV, clearly indicating that these responses were not JCV-specific. On the contrary, the VPl-p261 peptide shows 70% identity with the BKV homo log peptide, lower than that of the VP1- p36 and VP 1 -pi 00, which is of 89%> and 78%, respectively. Indeed these two latter peptides were shown to cross-react with BK virus (Krymskaya 2005, Li 2006, Sharma 2006, Tagaram 2008).
  • HLA Class I antigens with better binding with VPl-p261 were HLA-A*02 and A* 11, which are also the two most frequent alleles of the Caucasian population; however, the binding score was of 12, thus low (www.syfpeithi.de).
  • Low affinity between VPl-p261 and HLA-A*02 was confirmed by binding experiments, which showed fast association between VPl-p261 and HLA-A*02 molecule, but also rapid dissociation of the complex, suggesting that the peptide-MHC antigen complex has low stability and unlikely to work appropriately.
  • VPl-p261 Elispot strongly helps in the prognosis of the subsequent phases of PML and thus in the clinical management of the disease.
  • the test might be performed periodically (e.g., monthly or every second week) in a patient with PML: when using an Elispot assay, a SFU value above the cut-off level or an increased SFU value compared to previous responses may predict a favourable PML outcome (remission). Any other method to monitor T cell response may be used.
  • JCV VPl mutants are selected in PML patients that carry one of several aminoacid substitutions that confer a change in binding with their cell receptor.
  • two other commonly observed PML mutations are located within the authors' VPl l-p261 peptide: the 265D and the S267F/L/Y mutations. The authors thus hypothesised that a mutation at one of these positions does not only confer a change in binding affinity to cell receptor, but also affect the immune response directed against VPl .
  • the present invention provides a new tool (VPl-p261 and related assay, in particular an Elispot based assay) that is useful for real-time monitoring of JCV T-cell specific responses in PML patients or other patients, such as patient with multiple sclerosis or HIV+ patients.
  • VPl-p261 is highly immunogenic, i.e., it is recognised by more than two thirds of healthy controls, thus it would be an essential component of peptide pools that are to be applied to any individual to test T-cell responses for prognostic purposes.
  • JCV-specific T-cell responses may help predict patients who are more at risk of developing PML.
  • such approach may provide useful in patients who are going to initiate or who are receiving treatments with immunomodulatory molecules that have been associated with development of PML, such as natalizumab.
  • natalizumab treatment low or absent T-cell response would represent an additional variable to be used for PML risk stratification of patients (together with anti-JCV IgG, previous history of immune suppressive treatments and, in those already on natalizumab, duration of treatment, Gorelik 2010).
  • the authors disclosed a new possible mechanism of JCV evasion of the CTL response by selection of VPl mutations. This mechanism may provide an explanation to the frequently observed phenomenon by which JCV may propagate and lead to progressively fatal disease. More in general, these findings may help pave the way for new immune-based treatment approaches.

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Abstract

La présente invention concerne un peptide comprenant la séquence choisie dans le groupe : GMFTNRSGSQ (SEQ ID No.12), GMFTDRSGSQ (SEQ ID No.20), GMFTNRLGSQ (SEQ ID No.21), GMFTNRFGSQ (SEQ ID No.22), GMFTNRYGSQ (SEQ ID No.23) ou GMFTNRSGFQ (SEQ ID No.24) et son utilisation dans le diagnostic et/ou le pronostic de plusieurs pathologies comprenant la leucoencéphalopathie multifocale progressive.
PCT/EP2011/055448 2010-04-08 2011-04-07 Peptide immunodominant du virus du polyôme jc et son utilisation WO2011124652A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2548567A1 (fr) * 2011-07-22 2013-01-23 Universitätsklinikum Hamburg-Eppendorf Peptides du virus JC du polyome et protéines pour application de vaccination et de diagnostic
WO2013087601A3 (fr) * 2011-12-12 2013-08-08 Janssen Diagnostics Bvba Séquences peptidiques de polyomavirus
WO2014201034A3 (fr) * 2013-06-10 2015-03-12 Ansun Biopharma, Inc. Traitement de l'infection à polyomavirus
CN113913554A (zh) * 2021-11-08 2022-01-11 苏州药明检测检验有限责任公司 一种实时荧光定量pcr检测jc多瘤病毒的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GASNAULT JACQUES ET AL: "Critical role of JC virus-specific CD4 T-cell responses in preventing progressive multifocal leukoencephalopathy", AIDS, LONDON, GB, vol. 17, no. 10, 4 July 2003 (2003-07-04), pages 1443 - 1449, XP002520358, ISSN: 0269-9370, DOI: DOI:10.1097/00002030-200307040-00004 *
GLASS, A. J. AND VENTER, M.: "Improved detection of JC virus in AIDS patients with progressive multifocal leukoencephalopathy by T-antigen specific fluorescence resonance energy transfer hybridization probe real time PCR: evidence of diverse JC virus genotypes associated with progressive multifocal leukoencephalopathy in souther", JOURNAL OF MEDICAL VIROLOGY, vol. 81, 2009, pages 1929 - 1937, XP002640284 *
TESTA M ET AL: "JCV specific CTL responses against a novel HLA-A*02 restricted VP1 epitope", INFECTION, SPRINGER, vol. 38, no. Suppl. 1, 1 June 2010 (2010-06-01), pages 18, XP009149112, ISSN: 0300-8126 *
WEBER T ET AL: "PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY DIAGNOSED BY AMPLIFICATION OF JC VIRUS-SPECIFIC DNA FROM CEREBROSPINAL FLUID", AIDS, PHILADELPHIA,PA, US, vol. 8, no. 1, 1 January 1994 (1994-01-01), pages 49 - 57, XP000647605 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2548567A1 (fr) * 2011-07-22 2013-01-23 Universitätsklinikum Hamburg-Eppendorf Peptides du virus JC du polyome et protéines pour application de vaccination et de diagnostic
US9738690B2 (en) 2011-07-22 2017-08-22 Universitaet Zuerich Polyoma virus JC peptides and proteins in vaccination and diagnostic applications
WO2013087601A3 (fr) * 2011-12-12 2013-08-08 Janssen Diagnostics Bvba Séquences peptidiques de polyomavirus
WO2014201034A3 (fr) * 2013-06-10 2015-03-12 Ansun Biopharma, Inc. Traitement de l'infection à polyomavirus
US10300116B2 (en) 2013-06-10 2019-05-28 Ansun Biopharma, Inc. Treatment for BK polyomavirus infection
CN113913554A (zh) * 2021-11-08 2022-01-11 苏州药明检测检验有限责任公司 一种实时荧光定量pcr检测jc多瘤病毒的方法

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