METHOD FOR STRATIFYING THE RISK OF BK VIRUS NEPHROPATHY AFTER A KIDNEY TRANSPLANT

Abstract

The present application describes and claims a novel method for identifying and stratifying the risk of developing a BK virus nephropathy (“Nx BK-v” below) in patients having undergone a kidney transplant. This method uses an index combining at least three bio-markers: i) the intensity of the anti-BK-v memory T lymphocyte response (memory T lymphocytes which are specific to v, hereinafter “LTm anti-BK-v”), ii) the number of occurrences of incompatibility in the HLA alleles of class I and class II between the donor and the recipient of the graft, taking into account iii) the viral charge of the BK-v virus in the whole blood of the patient. The present method allows a very precise evaluation of the risk of developing an Nx BK-v during the months following the test with the aim of optimising the immuno-suppressive treatment in order to better preserve the transplanted kidney.

Claims

1. Method for assessing the risk of developing a BK virus (BKV) nephropathy in a patient who had kidney transplantation, said method comprising the following steps: a) measuring the viral load of BKV in a biological sample of said patient, b) measuring the normalized intensity of the CD4.sup.+ or CD8.sup.+ memory T cell response specific for BKV virus in a biological sample from said patient, said normalization being performed relative to the BKV viral load measured in step a), and c) determining the number of incompatibilities between the HLA I and II of the kidney donor and said patient.

2. Method according to claim 1, wherein the three parameters a), b) and c) are then compiled to create a stratification index for the risk that the patient will develop BK virus nephropathy.

3. Method according to claim 1, wherein the BKV viral load is measured in step a) by quantitative PCR from a sample of whole blood or plasma from said patient.

4. A method according to claim 1[[, 2 or 3]], wherein the response of the BKV specific CD4+ or CD8+ memory T cells measured in step b) is determined by contacting peripheral blood mononuclear cells from said patient with BKV virus peptides, and by assessing the proliferation of T cells present in said mononuclear cells after 4 to 7 days of culture, typically after 5 days of culture.

5. Method according to claim 1 [[4]], wherein the response of the BKV specific CD4+ or CD8+ memory T cells measured in step b) is determined by contacting peripheral blood mononuclear cells from said patient with BKV virus peptides, and by assessing the proliferation of T cells present in said mononuclear cells after 4 to 7 days of culture said proliferation is measured by determining the proportion of CD4+ T cells having diluted a proliferation marker such as CFSE after 4 to 7 days of culture in the presence of said peptides, typically after 5 days of culture.

6. Method according to claim 1, wherein the response of the BKV specific CD4.sup.+ or CD8.sup.+ memory T cells measured in step b) is determined by contacting peripheral blood mononuclear cells from said patient with BKV virus peptides, and by assessing the proliferation of T cells present in said mononuclear cells after 4 to 7 days of culture said proliferation is measured by determining the proportion of CD8.sup.+ T cells having diluted a proliferation marker such as CFSE after 4 to 7 days of culture in the presence of said peptides, typically after 5 days of culture.

7. Method according to claim 1, wherein a high risk of developing BK virus (BKV) nephropathy is concluded when: a) in relation with a viral load of 10.sup.3 copies of BK virus per mL of blood, b) the normalized intensity of the CD4.sup.+ or CD8.sup.+ memory T cell response specific for BKV is less than or equal to 100 intensity measurement units, and c) the number of incompatibilities between said patient and the donor of the transplanted kidney is less than or equal to 5.

8. Method according to claim 1, wherein a low risk of developing BK virus (BKV) nephropathy is concluded when: a) in relation with a viral load of 10.sup.3 copies of BK virus per mL of blood, b) the normalized intensity of the CD4.sup.+ or CD8.sup.+ memory T cell response specific for BKV is strictly greater than 100 intensity measurement units, and c) the number of incompatibilities between said patient and the transplant donor is strictly greater than 5.

9. In vitro method for assessing the response of a kidney transplant patient to blood replication of BKV after modification of immunosuppressive treatment, characterized in that the method as defined in claim 1 is repeated at regular time intervals after transplantation.

10. Method according to claim 1, wherein said method is an in vitro method for assessing the response of a kidney transplant patient to blood replication of BKV after modification of immunosuppressive treatment, and wherein said method is repeated before and after each treatment modification.

11. Method according to claim 1, wherein said method is an in vitro method for assessing the response of a kidney transplant patient to blood replication of BKV after modification of immunosuppressive treatment, and wherein said method is repeated approximately every 3 to 6 months.

12. Kit containing: a) reagents for conducting BK virus quantitative PCR, and b) reagents for conducting the immunological method described in claim 4 on whole blood.

13. Kit according to claim 12, wherein the reagents b) contain BKV peptide sequences or polyclonal lymphocyte stimulation peptide sequences and, optionally, lymphocyte labeling reagents.

14. Kit according to claim 12 containing: a) specific primers for amplifying portions of the BKV genome, and b) BKV peptide sequences, and, optionally c) the reagents necessary for conducting quantitative PCR with DNA extraction and/or d) the reagents for lymphocyte labeling.

Description

KEY TO FIGURES

[0202] FIG. 1 describes the frequency of BKV reactivation after kidney transplantation in terms of BKV viruria, isolated BKV viremia, BKV Nx and chronic dysfunction/loss of kidney transplants [4,5,6,7].

[0203] FIG. 2 describes the outcome at 3 years from the kidney transplant in patients with or without BKV Nx (a—kidney function assessed by measuring the glomerular filtration rate (GFR) in ml/min/1.73 m.sup.2, Kruskal-Wallis test and b—kidney transplant survival, Kaplan-Meier and log-rank test).

[0204] FIG. 3 shows the current approach for BKV Nx, based on assessment of the blood BKV viral load and kidney transplant biopsy.

[0205] FIG. 4 shows the method of the invention based on the combination of three parameters: [0206] i) a virological parameter assessed by the BKV viral load in the patient's blood, [0207] ii) an immunological parameter assessed by the intensity of the anti-BKV TCm response in the patient, and [0208] iii) a genetic parameter assessed by the number of HLA incompatibilities in the [donor/recipient] pair.

[0209] FIG. 5 shows the BKV viral load levels in the blood (a) and urine (b) of different patient groups of the cohort studied here (nonparametric Kruskal-Wallis test). The threshold used in the method of the invention (10.sup.3 copies/mL of BKV in the blood) was defined from these results in order to allow risk to be assessed as early as possible.

[0210] FIG. 6—part 1 shows the different lymphocyte functionality tests conducted after stimulation with specific BKV peptides. Cytokine secretion (1a) and lymphocyte proliferation (1b) were used to assess the anti-BKV TCm response in the different groups of patients of the cohort studied here (TC: T cells; nonparametric Kruskal-Wallis test).

[0211] FIG. 6—part 2 shows the different lymphocyte functionality tests conducted after stimulation by a mix of antiviral peptides (peptides covering cytomegalovirus, Epstein-Barr and influenza virus). Cytokine secretion (2a) and lymphocyte proliferation (2b) were used to assess the overall memory T cell antiviral response in the different groups of patients of the cohort studied here (no significant differences).

[0212] FIG. 7 shows the negative correlation between the intensity of the anti-BKV TCm response and the BKV viral load (copies/mL of blood) (nonparametric Spearman's correlation test).

[0213] FIG. 8/1 illustrates the assessment protocol for the anti-BKV TCm response, via the analysis of the proliferation of anti-BKV TCm in response to a pool of specific BKV peptides (proportion of CFSE.sup.low T cells). The proportion of anti-BKV TCm having actively proliferated (proportion of CFSE.sup.low TC) is measured by subtracting the proportion of CFSE.sup.low TC measured in the absence of peptide stimulation from the proportion of CFSE.sup.low TC measured in the presence of specific BKV peptides.

[0214] FIG. 8/2 shows the assessment of the normalized intensity of anti-BKV TCm response. The normalized intensity of anti-BKV TCm response is quantified in “normalized intensity measurement units”. A “normalized intensity measurement unit” is defined here as being the proportion of CFSE' anti-BKV TCm expressed for 10.sup.6 total T cells and normalized for 10.sup.3 copies of BKV per mL of blood. This normalization allows an intra and inter-individual comparison [here, inter-individual comparison (isolated BKV viremia vs. BKV Nx)].

[0215] FIG. 9 shows the normalized intensity of the anti-BKV CD8.sup.+ TCm cell response in patients with isolated BKV viremia (n=11) or BKV Nx (n=16). The only threshold used in the method of the invention (10.sup.2 normalized measurement intensity units) was defined from the results in order to take into consideration up to the 90.sup.th percentile of the response intensity values demonstrated in patients with BKV Nx.

[0216] FIG. 10 shows the comparison of the normalized intensity of the anti-BKV TCm response in type CD4 and CD8 lymphocytes in patients with isolated BKV viremia (n=11) and patients with BKV Nx (n=16).

[0217] FIG. 11a shows a lower number of HLA incompatibilities [HLA-A, -B, -DR, -DQ] between the transplant donor and recipient in patients with BKV Nx compared to patients with isolated BKV viremia (Mann-Whitney test). The threshold used in the method of the invention (5 HLA incompatibilities [HLA-A, -B, -DR, -DQ] between the transplant donor and recipient) was defined from these results in order to take into consideration up to the 75.sup.th percentile of the HLA incompatibility values in patients with isolated BKV viremia.

[0218] FIG. 11b shows the negative correlation between the number of HLA incompatibilities and BKV viremia (in number of copies/mL of blood) (nonparametric Spearman's correlation test).

[0219] FIG. 12 is a stratification tree for the BKV Nx risk according to the 3 parameters defined above.

[0220] FIG. 13 describes the level of risk of developing BKV Nx after kidney transplantation in cases of BKV viremia as a function of the following three parameters: [0221] i. the intensity of the anti-BKV TCm response normalized to, [0222] ii. the BKV blood viral load (10.sup.3 copies of BKV per mL of blood), and [0223] iii. the number of HLA incompatibilities between the donor and the recipient.

[0224] FIG. 14 shows the different indications for the method of the invention as well as the time interval for repeating said method.

[0225] FIG. 15 shows the approach proposed here for assessing BKV Nx risk, based on assessment of the blood BKV viral load and the method of the invention.

EXAMPLES

[0226] Materials and Methods:

[0227] In order to characterize the specific cellular response for BKV post-kidney transplantation, the present inventors conducted an observational study including 94 kidney transplant patients. The patients enrolled were all from the Nephrology Department of Bicetre Hospital (University Hospitals of South Paris). In all, this site performs more than 130 kidney transplants per year with an active file of more than 2000 patients monitored with a functional kidney transplant. The CPP Ile de France [ethics committee] was consulted and did not find any ethical obstacle to conducting this study. An information letter was given to all the patients and their consent was collected in writing.

[0228] This study started in November 2014 with a duration of enrollment of 36 months (enrollment finished on November 1, 2017). The inclusion criteria were the following: [0229] patients above age 18, [0230] no other organ transplantation except for the kidney one, [0231] no active chronic HIV/HBV/HCV infection.

[0232] Four groups of BKV virus reactivation were defined in these kidney transplant patients according to the criteria below: [0233] patients without BKV reactivation: [0234] plasma and urine BKV viral loads undetectable for at least 12 months. [0235] patients with BKV viruria: [0236] positive BKV viruria (>200 copies/ml) and [0237] BKV viremia undetectable for at least 12 months [0238] patients with isolated BKV viremia: [0239] positive BKV viremia (>200 copies/ml) for at last 6 months and [0240] no histological diagnosis of BKV Nx on kidney transplant biopsy [0241] patients with BKV Nx: [0242] diagnosis of BKV Nx on kidney transplant biopsy [0243] with positive BKV viremia (>200 copies/ml)

[0244] In order to characterize the anti-BKV TCm in the different groups of patients in this cohort, the present study included a clinical aspect (exhaustive collection of clinical and biological data over a prospective followup of 36 months) as well as an immunological aspect (study of peripheral blood mononuclear cells (PBMC) in a peripheral blood sample).

[0245] Regarding the clinical part of the study, the following clinical and biological data were collected as part of a longitudinal followup of 36 months: [0246] Age, sex, initial kidney disease, method and length of dialysis for the recipient [0247] Characteristics of the kidney transplantation: [0248] Age of the kidney transplantation [0249] Donor type [0250] Transplantation rank (first transplant or second/third/etc. transplant). [0251] Organ transplant rejection [0252] Reactivation of BKV virus: [0253] Blood and urine BKV viral loads (expressed in copies/mL) [0254] Duration of replication (expressed in months) [0255] Estimate of the glomerular filtration rate according to the MDRD formula: [0256] At 1 month and 6 months post transplant [0257] At diagnosis of BKV Nx or at 12 months post transplant [0258] At the end of followup [0259] Immunosuppressive treatment [0260] Induction treatment and maintenance treatment [0261] Residual amount of immunosuppressants [0262] at diagnosis of BKV Nx or at 12 months post kidney transplantation [0263] Immunosuppressive treatment changes following the diagnosis of BKV Nx.

[0264] Table 2 shows the absence of difference in terms of therapeutic immunosuppression (nature and intensity of immunosuppressive treatment) in the different groups of kidney transplant patients (NS=not significant).

TABLE-US-00002 TABLE 2 Immunosuppressive treatment in the different groups of kidney transplant patients (NS = not significant). Without BKV BKV BKV BK Plasma Immunosuppressive treatment reactivation viruria viremia exchanges NX P n 25 25 22 22 INDUCTION TREATMENT (n, %) 23 (92) 22 (88) 22 (100) 21 (95.5) NS Polyclonal depleting antibodies (n, %) 11 (47.8) 6 (27.3) 11 (50) 12 (57.1) NS Anti-CD25 monoclonal antibodies (n, %) 12 (52.2) 16 (72.7) 11 (50) 9 (42.9) MAINTENANCE TREATMENT before diagnosis of BKV nephropathy Tacrolimus (n, %) 18 (72) 19 (76) 15 (68.2) 17 (77.3) NS Residual amount of tacrolimus (ng/ml, 9.1 [8.1-11.4] 9.1 [8.4-10.4] 9.1 [7.7-11.1] 8.6 [6.6-10.1] NS median with interquartile) Antimetabolites (n, %) 23 (92) 23 (92) 15 (68.2 18 (81.8) NS Glucocorticoids (n, %) 25 (100) 25 (100) 22 (100) 22 (100) NS TRANSPLANT REJECTION TREATMENT before diagnosis of BKV nephropathy Intravenous glucocorticoid bolus (n, %) 5 (20) 5 (20) 8 (36.4) 10 (45.5) NS Polyclonal depleting antibodies (n, %) 1 (4) 1 (4) 3 (13.6) 1 (4.6) NS Plasma exchanges (n, %) 1 (4) 4 (16) 5 (22.7) 4 (18.2) NS RITUXIMAB (n, %) 2 (8) 2 (8) 4 (18.2) 2 (9.1) NS

[0265] Concerning the immunological part of the study, a single blood draw of 15 mL of whole blood (3 lithium heparin tubes of 5 mL) was performed at baseline. This blood draw was done during the histological assessments performed for the usual patient followup. In the groups with isolated BKV viremia and BKV Nx, a longitudinal followup was also conducted, with a new sample taken at 12 and 24 months from baseline in order to assess the progress of the anti-BKV TCm response. From 15 mL of whole blood, 8 to 10 million PBMC were isolated on average. This documents the relative lymphopenia of the patients (between 0.5 and 0.6 million PBMC/mL of blood).

[0266] The anti-BKV TCm response was characterized in the different patient groups. A specific activation of anti-BKV TCm was done by culturing PBMC with an overlapping peptide pool covering the BKV VP1 and LT-Ag proteins (PepTivator BKV, Miltenyi® or equivalent). These peptide pools activate CD4 and CD8 T cells and cover the two cycles of BKV replication (LT-Ag for the early phase and VP1 for the late phase of BKV virus replication). After activation by specific BKV peptides, the lymphocyte polyfunctionality of anti-BKV TCm was evaluated by flow cytometry in terms of: [0267] Cytokine secretions: [0268] overnight incubation of 4 million PBMC (800,000 PBMC/condition) in the presence of specific BKV proteins and brefeldin A (cytokine secretion inhibitor) then [0269] study of the IL2, IFN-γ and TNF-α. synthesis capacities. [0270] Lymphocyte proliferation: [0271] culture of 4 million PBMC labeled with CFSE (500,000 PBMC/condition) for 5 days in the presence of specific BKV peptides then [0272] study of the proliferation capacities by visualization of the CFSE dilution.

[0273] All the flow cytometry acquisitions were done in an 18-color cytometer (BD LSRFortessa™ or equivalent) and the analyses were done in FlowJo specific analysis software.

[0274] Statistical methodology used:

[0275] Continuous variables are expressed in median with interquartile range [25-75] given the absence of normal distribution of the data and the small number of subjects (n<30). These variables were compared by using Mann-Whitney or Kruskal-Wallis nonparametric tests according to the comparison of two or more distributions. The correlations were tested by using the nonparametric Spearman's correlation test.

[0276] The categorical variables are expressed in percentages. The chi-squared test was used to compare the proportions between the different groups. The survival functions were done according to the Kaplan-Meier estimator, with the log-rank comparison test.

[0277] The statistical significance threshold was established for a p-value<0.05 The statistical analysis was performed with GraphPad Prism software.

[0278] Development of the method of the invention:

[0279] a) Measurement of the Proliferation of Memory T Cells Specific for BKV

[0280] This lymphocyte functionality test based on the assessment of the anti-BKV TCm proliferation capacities after activation by BKV peptides was specifically developed by the present inventors.

[0281] FIG. 8/1 illustrates the protocol for identification of anti-BKV TCm that proliferated, in response to a specific stimulation by BKV virus peptides, from whole blood of kidney transplant patients.

[0282] PBMC comprising anti-BKV TCm were obtained from a 15-mL blood sample of whole blood for each patient. This blood sample is of the peripheral venous type, taken with 3 lithium heparin tubes (tubes with green caps) of 5 mL (FIG. 8/1a).

[0283] The proliferation test for anti-BKV TCm requires around 5 to 6 million PBMC; the blood sample provides 8 to 10 million.

[0284] The PBMC were isolated by FICOLL gradient C (PAN Biotech®—reference P04-60505 or equivalent). From 15 mL of whole blood, the present inventors isolate 8 to 10 million PBMC on average (FIG. 8/1b).

[0285] The PBMC were cultured for 5 days with two pools of BKV peptides at the final concentration of 1 μg/mL/peptide. The peptide pools are sold by Miltenyi Biotec (PepTivator® BKV VP1—reference: 130-097-272 and PepTivator® BKV LT—reference: 130-096-504) or equivalent (FIG. 8/1c). The LT-Ag and VP1 peptide sequences used are immuno-dominant peptides widely recognized in the scientific literature as able to activate anti-BKV TCm. The stronger of the two responses was considered for the identification of functional anti-BKV TCm.

[0286] The T cell proliferation was measured by addition, on the day of culture, of a lymphocyte proliferation marker (CellTrace CFSE Thermo Fisher Scientific®—reference: C34554). Other equivalent lymphocyte proliferation markers could also be used. On the fifth day of cell culture, the PBMC were labeled by the following antibodies [BD® anti-CD3 antibodies (reference: 564712), Miltenyi® CD4 (reference: 130-096-900) and Miltenyi® CD8 (reference: 130-096-561) or equivalent] and analyzed by flow cytometry (BD LSRFortessa cytometer or equivalent) (FIG. 8/1d). CD4 and CD8 TCm specific for BKV are identified and counted on the basis of the degree of CFSE labeling (CFSE.sup.low T cells). As explained above, the proportion of anti-BKV TCm having actively proliferated (proportion of CFSE.sup.low TC) is measured by subtracting the proportion of CFSE.sup.low TC measured in the absence of peptide stimulation from the proportion of CFSE.sup.low TC measured in the presence of specific BKV peptides (FIGS. 8/1e and 8/2a).

[0287] The proportion of functional anti-BKV TCm is then expressed for 10.sup.6 total T cells and then normalized for 10.sup.3 copies of BKV per mL of blood. FIG. 8/2 illustrates this normalization of the proportion of functional anti-BKV TCm in order to permit an assessment of the normalized intensity of the anti-BKV TCm response. The assessment of the intensity of this response permits an intra- and inter-individual comparison.

[0288] b) Measurement of Viral Load

[0289] The BKV viral load was measured via a specific commercial kit (BKV R-GENE®—reference 69-013B from BioMérieux® or equivalent). The biological sample used can be a whole blood or plasma sample from said patient.

[0290] c) Measurement of the Number of HLA Incompatibilities Between the Donor and the Recipient

[0291] All HLA typings in the context of transplantation are done by the St Louis HLA laboratory (which centralizes these tests on Ile de France. The HLA typing of the recipient is currently done by next-generation allelic sequencing techniques (allelic sequencing of HLA A, B, C, DRB1, DQB1, DQA1 and DPB1 molecules—MiSeq device from Illumina® or equivalent). HLA donor typing is done on an emergency basis, at the time of the kidney transplantation by a “Sequence Specific Primers method” (Linkage Biosciences, Thermo Fisher®) using SABR (Single Antigen Bead Resolution) kits or equivalent.

[0292] It is important to remember that donor and recipient HLA typing is routinely done according to the international recommendations for all kidney transplant patients at the time of the transplant in order to decide whether to proceed with the kidney transplantation. The results are transmitted to the medical teams via the Agence de la Biomédecine [French Biomedicine Agency].

[0293] Results:

[0294] Ninety-four kidney transplant patients were enrolled and divided into 4 groups according to their level of BKV virus reactivation: [0295] Patients without BKV reactivation (n=25): [0296] plasma and urine BKV viral loads <200 copies/mL [0297] Patients with BKV viruria (n=25): [0298] median urinary BKV viral load of 1.9×10.sup.4 [3.9×10.sup.3 −1.8×10.sup.5] copies/mL [0299] plasma BKV viral load (>200 copies/ml) [0300] median time until reactivation of 21.8 [14.6−34.7] months. [0301] Patients with isolated BKV (n=22): [0302] median plasma BKV viral load of 4.2×10.sup.3 [1.1×10.sup.3−1.2×10.sup.4] copies/mL [0303] urinary BKV viral load of 1.1×10.sup.8 [6.2×10.sup.6 −3.8×10.sup.8] copies/mL [0304] median time until reactivation of 10.3 [5.6 −21.1] months and 13.8 [9.3−27.1] months [0305] no histological diagnosis of BKV Nx on kidney transplant biopsy [0306] Patients with BKV Nx (n=22): [0307] histological diagnosis of BKV Nx on kidney transplant biopsy [0308] plasma BKV viral load of 2.8×10.sup.5 [3×10.sup.4−6.3×10.sup.5] copies/mL [0309] urinary BKV viral load of 1×10.sup.9[5.4×10.sup.7−1.8×10.sup.9] copies/mL [0310] respective median time until reactivation of 12.6 [5.2−24.7] months and 13.8 [4−23.3] months

[0311] FIG. 5 shows the different levels of blood and urinary BKV viral loads in the different patient groups as a function of the degree of BKV reactivation. In agreement with the prior art, it is important to note that while a plasma BKV viral load>10.sup.5 can be associated with BKV Nx (FIG. 5a), this parameter is late because it is already associated with renal parenchymal damage and severe chronic dysfunction of the transplant at the time of diagnosis of BKV Nx (FIG. 2a). Furthermore, given the absence of difference between the urinary BKV viral loads in patients with isolated BKV viremia and BKV Nx, this parameter was not considered in the development of the invention.

[0312] Based on the clinical and biological data obtained to date, the average time to transplant for the entire cohort studied was 3.5 years. The median time to onset of BKV Nx was 12 months after kidney transplantation.

[0313] Due to standardized single-center management of immunosuppression, the groups were comparable in terms of immunosuppression (nature and intensity of immunosuppressive treatment—see Table 2).

[0314] A chronic severe kidney transplant dysfunction was found from the diagnosis of BKV Nx (p<0.0001, FIG. 2a). The renal outcome was also unfavorable with a higher rate of transplant loss in patients with BKV Nx compared to patients without BKV Nx (p<0.0001, FIG. 2b).

[0315] An impaired functionality of anti-BKV TCm was documented in patients with BKV Nx post-kidney transplantation. The group of patients with BKV Nx exhibited a lower anti-BKV TCm response: [0316] in terms of cytokine secretion capacity: reduced interferon y secretion capacity in the BKV Nx group relative to the group with BKV viruria (FIG. 6/1a). [0317] in terms of lymphocyte proliferation capacity: reduced lymphocyte proliferation capacity in the BKV Nx group relative to the other three groups (patients without BKV reactivation, with BKV viruria and isolated BKV viremia (FIG. 6/1b).

[0318] This impairment of lymphocyte response was specific to anti-BKV TCm. Patients with BKV Nx possessed preservation of anti-viral responses after stimulation by a pool of peptides covering cytomegalovirus, Epstein-Barr and influenza viruses (CEF peptide pool from Axxora®, reference PT-PA-CEF-002 or equivalent—FIG. 6/2a-b).

[0319] The intensity of the anti-BKV TCm proliferative response was negatively correlated with the BKV viral load in the blood (FIG. 7). After normalization of the response intensity to 10.sup.3 copies of BKV/mL of blood, this normalized intensity of the anti-BKV response was significantly lower in patients with BKV Nx relative to patients with isolated BKV viremia (FIG. 9). These data suggest an alteration of the lymphocyte polyfunctionality of the anti-BKV TCm, suggesting a state of exhaustion of these lymphocytes.

[0320] Lymphocyte proliferation is the most discriminating assessment method for lymphocyte functionality. Moreover, this test allows specifically assessing central memory T cells and memory stem cells. However, as explained previously, the size of these lymphocyte subpopulations within the memory T cells specific to a pathogen determines their capacities for self-renewal, proliferation, help signals and differentiation into effectors.

[0321] The specific assessment of these central memory and memory stem cells permits a more precise assessment of the immunological capacities for protection against BKV virus. The assessment of the cytokine secretion capacity makes it possible to preferentially study “terminal” memory cell subpopulations of the memory effector or terminal effector T cell type. These highly differentiated lymphocyte subpopulations have little, if any, self-renewal, proliferation or differentiation capacity ([13], [14]). This assessment method was therefore favored.

[0322] Moreover, as mentioned above, a smaller number of HLA incompatibilities has been documented in patients with BKV Nx. Patients with BKV Nx had a lower total number of HLA incompatibilities [HLA-A, -B, -DR, and -DQ] relative to patients with isolated BKV viremia (FIG. 11a). Moreover, this number of incompatibilities was negatively correlated to the BKV viral load (FIG. 11b).

[0323] From the data obtained, a stratification index of the risk of developing BKV Nx was developed.

[0324] In patients with BKV Nx, it was possible to document an association between: [0325] a reduction in the normalized intensity of anti-BKV TCm response (intensity of response normalized for 10.sup.3 copies/mL of blood of BKV virus) and [0326] a low number of HLA molecule incompatibilities between the donor and the recipient.

[0327] Thus, in cross-sectional analysis of the cohort, 100% of patients with a normalized intensity of the anti-BKV TCm response≤10.sup.2 and a total number of HLA incompatibilities≤5 had BKV Nx (FIG. 13, dark gray dial). Conversely, no patient having a normalized intensity of the anti-BKV TCm response>10.sup.2 and a total number of HLA incompatibilities >5 had BKV Nx (FIG. 13, white dial).

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