Use of at least one biomarker for the in vitro prognosis or diagnosis of lymphoproliferative episodes associated with the Epstein-Barr virus (EBV)

Abstract

A protein complex isolated from its natural medium and including the ZEBRA protein of sequence SEQ ID NO: 1, the isolated protein complex having the following properties: it is more stable than the ZEBRA protein, in particular it is more resistant to the action of the proteases than the ZEBRA protein, it is capable of specifically binding the AZ125 and AZ130 monoclonal antibodies, it is soluble in a body fluid, and in particular a body fluid selected from the group constituted by blood and serum,
and to its detection process and the uses of this process for the prognosis or diagnosis of lymphoproliferative episodes associated with the EBV.

Claims

1. A method of diagnosing malignant lymphoproliferation associated with a replicative form of Epstein-Barr virus (EBV) or with a tumor associated with EBV, in a patient seropositive for EBV, said process comprising: providing a biological body fluid sample collected from said patient seropositive for EBV; and conducting a sandwich ELISA on said sample to measure the amount of a soluble circulating protein complex comprising BamHI Z Epstein-Barr virus replication activator (ZEBRA) protein of sequence SEQ ID NO: 1, wherein in said sandwich ELISA, AZ125 antibody is absorbed on a suitable plate to capture the soluble circulating protein complex, and AZ130 antibody is used for detecting the protein complex captured by the AZ125 antibody; wherein a measured amount of said soluble circulating protein complex greater than or equal to 0.15 micrograms/ml of biological body fluid indicates said patient having said malignant lymphoproliferation.

2. The method according to claim 1, wherein the patient is an immunosuppressed patient or immunodeficient patient belonging to the group consisting of patient awaiting a transplant, patient having received a transplant, patient seropositive for an immunosuppressive virus, patient seropositive for HIV, and patient having a genetically transmitted immunological deficit.

3. The method according to claim 2, for the diagnosis of a first episode of lymphoproliferation, said first episode of lymphoproliferation occurring before initiation of any therapeutic treatment of a lymphoma.

4. The method according to claim 2, for the diagnosis of a reactivation of the replicative form of EBV occurring following a treatment of the patient for a first episode of lymphoma associated with EBV reactivation.

5. The method according to claim 2, wherein the patient is immunocompetent and does not present clinical signs of infectious mononucleosis.

6. The process according to claim 1, wherein said biological body fluid sample is blood or serum.

7. The process according to claim 1, wherein said tumor associated with EBV is nasopharyngeal cancer.

8. A method of measuring the amount of a soluble circulating protein complex comprising the BamHI Z Epstein-Barr virus replication activator (ZEBRA) protein of sequence SEQ ID NO: 1 in a biological body fluid sample, the method comprising: providing the biological body fluid sample; and conducting a sandwich ELISA on said sample, wherein in said sandwich ELISA, AZ125 antibody is absorbed on a suitable plate to capture the soluble circulating protein complex, and AZ130 antibody is used for detecting said protein complex captured by the AZ125 antibody.

Description

LEGEND OF THE FIGURES

(1) FIG. 1: Diagrammatic representation of the different domains of the ZEBRA protein. The protein sequence of the ZEBRA protein numbers 245 amino acids. The different fields of the protein are represented by the blocks: A: activation domain, which extends from amino acids 1 to 140, B: DNA binding domain, which extends from amino acids 176 to 194, C: dimerization field, which extends from amino acids 195 to 221.
The arrows indicate the antigenic epitopes of the ZEBRA protein specifically recognized by the AZ125 and AZ130 antibodies.

(2) The present invention is illustrated by the following examples.

EXAMPLE 1: ASSAY OF THE CIRCULATING ZEBRA ANTIGEN AND ANTI-ZEBRA ANTIBODIES IN NON-IMMUNOSUPPRESSED PATIENTS

(3) 1Patients

(4) A cohort of 14 non-immunosuppressed patients (MST Consultations), seropositive for EBV but having no PTLD were tested and the presence of the ZEBRA antigen or anti-ZEBRA antibodies was assessed.

(5) 2Method of Assay of the ZEBRA Antigen

(6) The technique for detecting the soluble ZEBRA antigen is based on the five-stage ELISA method: (i) coating of a 96-well plate with the AZ 125 antibody (purified, diluted in PBS to a final concentration of 10 g/ml final). The plate is then placed overnight at 4-8 C. (ii) The plate is washed three times using a solution of PBS containing 0.5% Tween 20, followed by the saturation of the non-specific sites of the wells by adding saturation buffer (PBS-Tween 20 0.05%-FCS 10%-BSA 2%) for 60 minutes at ambient temperature under stirring. The plate is again rinsed three times. (iii) 100 l of serum previously diluted to 1/10 in dilution buffer (PBS-Tween 20 0.05%-FCS 10%) are added twice. In parallel, a standard range of the ZEBRA protein is produced. (iv) After washing, 50 l of the biotinylated AZ 130 antibody is added simultaneously at a concentration of 5 g/ml. The plate is incubated under stirring at ambient temperature for 2 hours. After three washings, the detection is carried out with alkaline phosphatase avidin (Invitrogen) diluted to 1/500 in PBS-Tween 20 0.05%-BSA 1%. Incubation takes place for 60 minutes at ambient temperature under stirring. The wells are then washed four times with the same buffer, PBS Tween 20 0.1%. (v) Development is carried out using a chromogenic substrate, p-nitrophenylphosphate, (Sigma). Thus the pNPP is presented in the form of tablets that must be diluted in diethanolamine buffer (pH=9.5) in order to obtain a concentration of 1 mg/ml. 100 l/well of this solution are added. The reaction must develop for 45 nm in darkness. The OD reading takes place at 405 nm (reference absorbance at 630 nm).

(7) 3Method of Assay of the Anti-ZEBRA Antibodies

(8) The assay of the anti-ZEBRA antibodies is carried out according to an ELISA test described by Marechal et al., 1993 Res. Virol. 144: 397-404. The recombinant ZEBRA protein originates from a strain of Escherichia coli transformed by the pET3c plasmid containing the coding part of the BZLF1 gene under the control of a transcription unit of the T7 polymerase type. The recombinant ZEBRA protein is used to coat the bottom of the wells of a microplate and serves for attaching the anti-ZEBRA antibodies. The anti-ZEBRA antibody titre is defined using the final point dilution method described by Vaur et al. 1986, J. Clin. Microbiol., 24: 596-599.

(9) 4Results

(10) The measurement of the concentration of the ZEBRA antigen as well as the titration of the anti-ZEBRA antibodies were carried out on samples of serums originating from the 14 patients of the cohort and were tested according to the techniques specified above. The results are shown in Table 1 below.

(11) TABLE-US-00001 TABLE 1 Detection of the ZEBRA antigen and anti-ZEBRA antibodies in the serum of immunocompetent patients. Anti-ZEBRA ZEBRA antigen IgG Patients (g/ml) (dilution) #80 <0.15 512 #81 <0.15 256 #82 <0.15 512 #83 <0.15 128 #84 <0.15 128 #87 <0.15 64 #88 <0.15 1024 #89 <0.15 16384 #91 <0.15 1024 #92 <0.15 64 #93 <0.15 128 #94 <0.15 <64 #95 <0.15 256 #96 <0.15 256

(12) The ZEBRA antigen is found at a concentration less than 0.15 g/ml in the serum of the 14 patients tested. The presence of anti-ZEBRA antibodies is detected in titres that can vary greatly from one patient to the other. In patients #88, #89 and #91, the EBV replication phase was more active than in patients #87 and #92. It is generally acknowledged that, starting from an anti-ZEBRA antibody titre of 128, EBV is in replication phase.

(13) These results show that the 14 immunocompetent patients presenting no PTLD of the cohort are all seropositive for the EBV virus but do not present strong replication of the virus measurable by the quantity of circulating ZEBRA antigen.

EXAMPLE 2: ASSAY OF THE CIRCULATING ZEBRA ANTIGEN, ANTI-ZEBRA ANTIBODIES AND THE EBV VIRAL LOAD IN IMMUNOSUPPRESSED PATIENTS PRESENTING NO PTLD

(14) 1Patients

(15) Three patients seropositive for EBV and having received a bone marrow transplant but presenting no clinical sign of lymphoproliferation were tested and the presence of the ZEBRA antigen or anti-ZEBRA antibodies as well as the measurement of the EBV viral load were assessed.

(16) 2Method of Assay of the ZEBRA Antigen and Anti-ZEBRA Antibodies

(17) The methods have been described precisely in Example 1 above.

(18) 3Measurement of the EBV Viral Load

(19) 3.1Extraction of the DNAs

(20) Total DNA extraction is carried out using a MagNA Pure Compact Nucleic Acid Isolation kit (Roche-Applied). In brief, the mononucleated cells (PBMC) are isolated by centrifugation on Ficoll-Hypaque, density 1.077, from a sample of whole blood collected on EDTA. The PBMCs are lysed with a solution containing chaotropic salts and proteinase K and the nucleic acids are captured on the surface of magnetic beads (Glass Magnetic Particles). Successive washings make it possible to elute the non-bound substances and the DNA is eluted with a saline buffer at a low concentration.

(21) 3.2Standard Ranges

(22) Three standard ranges are established from a series of dilutions of plasmid constructions comprising PCR products relating to the BALF-5 or ZEBRA genes. A double-stranded DNA of 90 pb for the BALF-5 gene and 100 pb for the ZEBRA gene were each cloned in a pCR2.1 vector.

(23) The BALF-5 and ZEBRA standard ranges extend from 10.sup.3 to 10.sup.8 copies/ml.

(24) The slopes of the standard ranges are respectively: 3.47 with an intercept at 47.42 and an R.sup.2 value of 0.997869 for BALF-5, 3.41 with an intercept at 46.62 and an R.sup.2 value of 0.998847 for ZEBRA (probe of SEQ ID NO: 7), and 3.49 with an intercept at 47.42 and an R.sup.2 value of 0.997320 for ZEBRA (probe of SEQ ID NO: 8).
These results demonstrate the perfect linearity of these two standard ranges. The PCR effectiveness is 94% for BALF-5 and 97% (probe of SEQ ID NO: 7) or 93% (probe of SEQ ID NO: 8) for ZEBRA respectively.

(25) 3.3PCR

(26) The pair of primers and the probe specific to the BALF-5 gene encoding the DNA polymerase of EBV have already been described in the literature (Kimura et al., J. Clin. Microbiol. 1999 January; 37(1): 132-136).

(27) A pair of primers and a probe have been specifically designed by the inventors for ZEBRA. The sequences are given in Table 2 below:

(28) TABLE-US-00002 TABLE2 Sequencesoftheprimersandprobesusedfor thedetectionoftheEBVviralload. Balf-5(F) SEQIDNO:2 5-CGGAAGCCCTCTGGAC TTC-3 Balf-5(R) SEQIDNO:3 5-CCCTGTTTATCCGATG GAATG-3 Balf-5(S) SEQIDNO:4 5-FAMTGTACACGCACG AGAAATGCGCCTAMRA-3 ZEBRA(F) SEQIDNO:5 5-CCAGGCTTGGGCACAT CT-3 ZEBRA(R) SEQIDNO:6 5-CCCATCTAAACGCCTG ATTTTT-3 ZEBRA(S) SEQIDNO:7 5-FAMCATTTTCAGATG ATTTGGCAGCAGCCAC TAMRA-3 ZEBRA(S) SEQIDNO:8 5-FAMCTTCAACAGGAG GCGCTAMRA-3

(29) The BALF-5 reaction mix comprises 5 pmol of probe of sequence SEQ ID NO: 4, 7.5 pmol of sense primer of sequence SEQ ID NO: 2, 7.5 pmol of anti-sense primer of sequence SEQ ID NO: 3, 1 of Taq Man Universal PCR Master Mix (Applied Biosystems) and 10% of Pre-Mix Cl Simplexa diluted to 1/100.

(30) The ZEBRA reaction mix comprises 5 pmol of probe of sequence SEQ ID NO: 7, 7.5 pmol of sense primer of sequence SEQ ID NO: 5, 7.5 pmol of anti-sense primer of sequence SEQ ID NO: 6, 1 of Taq Man Universal PCR Master Mix (Applied Biosystems) and 10% Cl Simplexa Pre-Mix diluted to 1/100.

(31) The amplification thermal profile is: 1 cycle of 2 minutes at 50 C., 1 cycle of 10 minutes at 95 C., 45 cycles of [15 seconds at 95 C. then 1 minute at 60 C.].

(32) The amplifications are carried out on an ABI Prism 7500 device (Applied Biosystems).

(33) The size of the amplicons obtained is 90 pb for Balf-5 and 100 pb for ZEBRA respectively.

(34) 4Results

(35) The measurement of the concentration of the ZEBRA antigen, the titration of the anti-ZEBRA antibodies and the measurement of the EBV viral load were carried out on samples of serums originating from 3 patients were tested according to the techniques specified above. The results are shown in Table 3 below.

(36) TABLE-US-00003 TABLE 3 Detection of the ZEBRA antigen, anti-ZEBRA antibodies and the EBV viral load in theserum of immunosuppressed patients not developing PTLD. Anti- EBV viral ZEBRA ZEBRA load antigen IgG Patients Date (copies/ml) (g/ml) (dilution) #1 = AK Jan. 12, 2010 transplant Feb. 15, 2010 0 <0.15 1024 Apr. 23, 2011 925 <0.15 1024 #2 = BER Sep. 23, 2009 transplant Oct. 12, 2009 0 <0.15 64 Dec. 09, 2009 0 <0.15 512 #3 = AUG Sep. 15, 2009 transplant Oct. 05, 2009 0 <0.15 256 Dec. 28, 2009 7210 <0.15 256

(37) For patients #1 and #3, the EBV viral load, which is undetectable during the month following the transplant, reaches approximately 10.sup.3 to 10.sup.4 copies/ml one hundred days after the transplant has taken place. In parallel, the quantity of anti-ZEBRA antibodies does not vary during this time interval. Finally, the ZEBRA antigen is at a concentration less than 0.15 g/ml, which means that EBV is not in strong replication phase. Despite increasing the viral load, it therefore appears unnecessary to treat these patients in order to prevent a lymphoma.

(38) For patient #2, the viral load remains at zero and the ZEBRA antigen is at a concentration less than 0.15 g/ml not only during the month following the transplant but also 77 days after the transplant has taken place. On the other hand, the patient has an anti-ZEBRA antibody titre comprised between 64 and 512 which indicates not only that the patient is seropositive for EBV but that the virus is very probably starting strong replication in the host cells.

EXAMPLE 3: ASSAY OF THE CIRCULATING ZEBRA ANTIGEN, ANTI-ZEBRA ANTIBODIES, EBV VIRAL LOAD AND IL-10 IN IMMUNOSUPPRESSED PATIENTS PRESENTING A PTLD

(39) 1Patients

(40) Four patients who are seropositive for EBV, having received a bone marrow transplant and for whom lymphoproliferation was diagnosed were studied. The presence of the ZEBRA antigen or anti-ZEBRA antibodies as well as the measurement of the EBV viral load and the concentration of IL-10 were assessed. A kinetic study of these biological parameters was carried out.

(41) 2Method of Assay of the ZEBRA Antigen and Anti-ZEBRA Antibodies

(42) The methods are described precisely in Example 1 above.

(43) 3Measurement of the EBV Viral Load

(44) The methods are described precisely in Example 2 above.

(45) 4IL-10 Assay

(46) IL-10 assay of the samples of patients is carried out using the commercial kit Human IL-10 CytoSet (Invitrogen, Camarillo, USA) according to a five-stage ELISA method: (i) coating of a 96-well microplate with anti-Human IL-10 antibodies at a rate of 0.1 g/well. The microplate is incubated at 4 C. for from 12 to 18 hours. (ii) washing of the microplate then incubation of 300 l/well of Assay Buffer solution at ambient temperature for one hour. (iii) addition of 100 l of standard or of samples and 50 l of detection antibodies coupled with biotin/well and incubation under stirring at ambient temperature for 2 hours. (iv) the microplate is rinsed 5 times and 100 l of streptavidin-HRP/well is incubated under stirring at ambient temperature for 30 minutes. (v) the microplate is rinsed 5 times and 100 l of TMB chromogenic substrate/well is incubated under stirring at ambient temperature for 30 minutes. At the end of this incubation period 100 l of stopping solution is added to each of the wells. OD reading takes place at 450 nm during the 30 minutes following the addition of the stopping solution.

(47) 5Results

(48) The measurement of the concentration of the ZEBRA antigen, the titration of the anti-ZEBRA antibodies, the measurement of the EBV viral load and the IL-10 assay were carried out on samples of serums originating from the 4 patients according to the techniques specified above. The results are shown in Table 4 below.

(49) TABLE-US-00004 TABLE 4 Detection of the ZEBRA antigen, anti-ZEBRA antibodies, EBV viral load and concentration of IL-10 in the serum of immunosuppressed patients developing a PTLD. Anti- EBV viral load ZEBRA ZEBRA (copies/150,000 antigen IgG IL-10 cells) (g/ml) (dilution) (pg/ml) Patient # 1 D0 Transplant D21 0 <0.15 <64 0 D36 2.5 10.sup.4 <0.15 <64 0 D56 2.0 10.sup.6 8.49 256 0 D61 1.0 10.sup.4 1.62 0 0 D68 1.2 10.sup.4 <0.15 64 0 D71 1.7 10.sup.4 <0.15 <64 100 D75 3.9 10.sup.4 <0.15 64 0 D82 1.6 10.sup.4 7.61 64 0 D89 1.5 10.sup.3 <0.15 64 0 D93 6.9 10.sup.3 <0.15 128 0 D96 1.2 10.sup.3 <0.15 64 0 D103 1.9 10.sup.3 <0.15 256 0 D110 1.7 10.sup.3 1.54 ND 0 D124 0 8.32 64 ND Patient # 2 D0 Transplant D88 0 96.5 512 220 D102 5.7 10.sup.3 36.17 <64 150 D141 1.7 10.sup.3 <0.15 <64 0 D144 2.9 10.sup.3 <0.15 <64 120 D151 6.3 10.sup.3 <0.15 64 0 D159 6.0 10.sup.5 <0.15 64 150 Patient # 3 D0 Transplant D11 5.3 10.sup.2 <0.15 128 70 D14 1.9 10.sup.3 <0.15 512 70 D18 1.7 10.sup.4 4.59 <64 0 D32 ND 1.71 ND ND D39 7.5 10.sup.4 <0.15 256 0 Patient # 4 DO Transplant D14 0 <0.15 128 ND D21 ND <0.15 ND ND D29 ND <0.15 ND ND D32 ND <0.15 ND ND D35 1.3 10.sup.4 7.48 1024 ND D38 ND <0.15 ND ND D42 1.2 10.sup.4 <0.15 512 ND D63 ND <0.15 ND ND D72 ND <0.15 ND ND D80 2.1 10.sup.4 <0.15 512 ND D95 ND <0.15 256 ND D102 1.2 10.sup.4 <0.15 256 ND D112 ND 23.24 ND ND D121 ND 23.65 ND ND D135 ND 1.62 ND ND D143 ND 3.14 ND ND
Patient #1:

(50) Three weeks after the transplant has taken place, all the biological markers tested are at their lowest.

(51) On D36, only the EBV viral load varies in order to reach 2.5.Math.10.sup.4 copies/150,000 cells. On D56, the EBV viral load reaches 2.Math.10.sup.6 copies/150,000 cells, the ZEBRA antigen is detected at 8.49 g/ml and the anti-ZEBRA antibody titre has increased to 256. These markers are the clinical sign of a strong EBV replication evidenced by the ZEBRA antigen peak, the rise in the anti-ZEBRA antibody level in combination with an increase in the pool of B lymphocytes infected with EBV (EBV viral load). PTLD is diagnosed on D56 and the patient is treated with Mabthera once a week for four weeks.

(52) From D61 to D75, the EBV viral load, the ZEBRA antigen level and the quantity of anti-ZEBRA antibodies decrease and stabilize at values close to those found on D36. This means that the viral replication has dropped. However on D71 an IL-10 synthesis peak is observed.

(53) On D82, the ZEBRA antigen is detected at a value of 7.61 g/ml whilst neither the EBV viral load nor the anti-ZEBRA antibody titre have been modified with respect to D68. This increase in the ZEBRA antigen concentration indicates that there is a resumption of the strong EBV replication.

(54) While the EBV viral load changes little between D89 and D110, the anti-ZEBRA antibody titre increases with peaks on D93 and on D103.

(55) On D110, the concentration of the ZEBRA antigen increases to 1.54 g/ml, a sign of a new strong replication phase of EBV. At the same time, a new episode of PTLD is diagnosed. The patient is treated with chemo- and radiotherapy before receiving a second transplant on D124. These treatments result in a drop in the anti-ZEBRA antibody titre.

(56) Patient #2:

(57) On D88, whilst the EBV viral load is zero, the concentration of ZEBRA antigen reaches 96.5 g/ml, the anti-ZEBRA antibody titre is 512 and the IL-10 is at 220 pg/ml. These markers are the sign of a strong EBV replication.

(58) On D102, the EBV viral load reaches 5.7.Math.10.sup.3 copies/150,000 cells, a sign that a greater number of B lymphocytes are infected with EBV.

(59) On D141 and D144, although the EBV viral load remains stable, it is possible to observe not only a reduction in the anti-ZEBRA antibody titre but also a reduction in ZEBRA antigen concentration. This reflects a drop in the replicative activity of EBV. In parallel on D144, an IL-10 secretion peak is observed.

(60) On D159, the EBV viral load increases by two logs and a new IL-10 peak is detected. PTLD is diagnosed and the patient is treated with Mabthera.

(61) Patient #3

(62) Two weeks after the transplant has taken place (D14), the anti-ZEBRA antibody titre increases in order to reach 512. This reflects the start of strong EBV replication. This hypothesis is confirmed on D18 by the increase in the ZEBRA antigen level which passes from <0.15 g/ml to 4.59 g/ml and by the increase in the EBV viral load by one log in four days.

(63) On D39, PTLD is diagnosed.

(64) Patient #4

(65) On D35, the EBV viral load reaches 1.3.Math.10.sup.4 copies/150,000 cells, the ZEBRA antigen is detected at 7.48 g/ml and the anti-ZEBRA antibody titre has increased to 1024. These markers are the clinical sign of a strong EBV replication evidenced by the ZEBRA antigen peak, the rise in the anti-ZEBRA antibody level in combination with an increase in the pool of B lymphocytes infected with EBV (EBV viral load). PTLD is diagnosed on D35 and the patient is treated with Mabthera once a week for four weeks.

(66) As from D112, the ZEBRA antigen level varies from 23 to 1.62 g/ml, suggesting a resumption of the strong EBV replication.