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Department of Public Health and Infectious Diseases, “Sapienza” University, P.le Aldo Moro, 5, 00185 Rome, ItalyIRCSS San Raffaele Pisana, Microbiology of Chronic Neuro-degenerative Pathologies, Rome, Italy
IRCSS San Raffaele Pisana, Microbiology of Chronic Neuro-degenerative Pathologies, Rome, ItalyDepartment of Public Health and Infectious Diseases, Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza University of Rome, Italy
Anti-JCPyV antibody assays are not sufficient to detect a JCPyV infection.
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JCPyV-DNA detection to identify JCPyV+ patients with antibodies not yet detectable.
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NCCR re-organizations as viral biomarkers correlated with neuro-virulent variants.
Abstract
Background
The risk of progressive multifocal leukoencephalopathy (PML), a brain infection caused by John Cunningham virus (JCPyV), is the main limitation to the use of natalizumab, highly effective in the treatment of relapsing remitting multiple sclerosis (RRMS) patients. Establishing the PML risk against expected benefits represents an obligatory requirement of MS treatment algorithm. In order to achieve this goal, the aims of this study were to establish if JCPyV-DNA detection and non-coding control region (NCCR) arrangements could play a role of biomarkers, supporting anti-JCPyV antibodies measurement, actually the only parameter for PML risk stratification.
Methods
Thirty RRMS patients in treatment with natalizumab were enrolled. Urine and blood samples were collected according to this calendar: baseline (T0), 4 (T1), 8 (T2), 12 (T3), 16 (T4), 20 months (T5) after beginning of natalizumab therapy. After JCPyV DNA extraction, a specific quantitative-PCR (Q-PCR) and arrangements’ analysis of NCCR and Viral Capsid Protein 1 (VP1) were carried out.
Results
Q-PCR detected JCPyV DNA in urine and blood from baseline (T0) to 20 natalizumab infusions (T5), although JC viral load in urine was significantly higher compared to viremia, at all selected time points. A contextual analysis of the anti-JCPyV-antibodies versus JCPyV-DNA detection revealed that viral DNA preceded the antibodies’ presence in the serum. During the first year of natalizumab treatment, sequences isolated from blood displayed an archetype JCPyV NCCR structure with the occurrence of point mutations, whereas after one year NCCR re-organizations were observed in plasma and PBMC with duplication of NF-1 binding site in box F, duplication of box C and partial or total deletion of box D. VP1 analysis showed the amino acid change mutation S269F in plasma and S267L in PBMC, involving the receptor-binding region of VP1. Phylogenetic analysis suggested a stability and a similarity across different isolates of the JCPyV VP1.
Conclusions
We highly recommend considering JCPyV-DNA detection and NCCR re-organizations as viral biomarkers in order to accurately identify JCPyV-infected patients with a specific humoral response not yet detectable and to identify NCCR arrangements correlated with the onset of neurovirulent variants.
The arsenal of medications for the treatment of multiple sclerosis (MS), a complex chronic, autoimmune, demyelinating disease affecting central nervous system (CNS) (
), has been enriched by a variety of substances with differing mechanisms of action. Specifically, to date, 13 disease-modifying therapies (DMTs) for treatment of relapsing-remitting multiple sclerosis (RRMS), the most prevalent MS phenotype, have been approved by the Food and Drug Administration (FDA) and have changed the scenery for care of RRMS patients (
), they are also correlated with a higher risk of serious side effects. Several DMTs have been associated with progressive multifocal leukoencephalopathy (PML), a demyelinating disease of CNS, caused by John Cunningham virus (JCPyV) (
). PML evolves in the context of an underlying immunological anomaly and in cell-mediated immunity deficiency, as demonstrated by more than 60% of PML cases occurred in association with hematological malignancies (
the most potent effective DMT for relapse prevention, is a monoclonal antibody which prevents the linkage between α4β1 and its ligand (vascular cell adhesion molecule-1) blocking transmigration of activated lymphocytes into CNS (
), undergoing deletion and enhancement processes, could generate arranged NCCR (rr-NCCR) sequences, observed during host's immunosuppression and correlated with a poor prognosis in PML patients.
In the last two decades, several studies have been carried out in order to identify markers which could improve safety and efficacy of natalizumab therapy, nevertheless PML cases were reported (
). As of May 2016, the global incidence of PML during natalizumab treatment was 4.22 per 1000 patients. (https://medinfo.biogen.com/). Three factors stratify risk: natalizumab therapy > 2 years, an antibodies index for JCPyV > 1,5 and previous immunosuppressive therapy (
). Actually, anti-JCPyV antibodies index is the only parameter for PML risk stratification, although many others candidates, such as L-selectin (CD62L) expression, T-cell response to JCPyV reactivation in PML, microRNAs expression during natalizumab therapy (
), establishing risk against expected benefits represents an obligatory requirement of MS treatment algorithm. In order to achieve this goal, this study was conducted on a RRMS cohort of patients in treatment with natalizumab to establish if JCPyV-DNA detection in different body compartments and NCCR arrangements could play a role of biomarkers, supporting anti-JCPyV antibodies measurement and contributing to PML risk monitoring and assessment in the individual-patient during therapy. Moreover, to further understand if natalizumab might affect JCPyV strains circulating in our cohort, a Viral Capsid Protein 1 (VP1) alignment and a phylogenetic analysis were performed.
2. Methods
2.1 Study participants and samples collection
After signed informed consent based on the approved Ethic Committee of Policlinico Umberto I of Rome (protocol number 130/13), a cohort of 30 RRMS subjects, followed up at the Department of Human Neurosciences of Sapienza University of Rome, was recruited between March 2016 and March 2018. All participants fulfilled the Italian Agency of Drug (AIFA) criteria for natalizumab treatment. Therapeutic protocol consisted of administration of 300 mg intravenous natalizumab every 4 weeks. The enrolled patients (12 males/18 females, mean age ± stand. dev. 30.2 ± 6.6; mean months of illness ± stand. dev. 85 ± 85.5; mean Expanded Disability Status Scale (EDSS) ± stand. dev. 1.9 ± 1.3) were visited before natalizumab treatment (baseline: 0 infusions) and every 4, 8, 12, 16, 20 infusions. Demographic and clinical characteristics are presented in Table 1.
Table 1RRMS patients’ demographic and clinical characteristics.
Variables
RRMS (n = 30)
Age years (mean, SD)
30.2 ± 6.6
Sex, female (n) and male (n)
female (18) and male (12)
Mean months of illness years (SD)
85 ± 85.5
Mean EDSS (SD)
1.9 ± 1.3
Therapy before starting natalizumab
No therapy (/n)
12/30
Interferon β (/n)
15/30
Mitoxantrone and interferon β (/n)
2/30
Glatiramer acetate (/n)
1/30
RRMS: Relapsing-remitting multiple sclerosis; N: Number; SD: Standard deviation; EDSS: Expanded disability status scale.
Samples were collected according to this calendar: baseline (T0), 4 (T1), 8 (T2), 12 (T3), 16 (T4), 20 months (T5) after beginning of therapy. In detail, 30 plasma and 30 peripheral blood mononuclear cells (PBMC) (from whole blood in EDTA) and 30 urine samples were obtained for a total of 540 specimens.
After the activation of the STRATIFY JCVⓇ service (Biogen Idec), assessment of anti-JCPyV antibodies (IgG) on serum of the enrolled subjects was performed at T0 and after 1 year from the start of the follow-up (T3) (
). At each follow-up visit, disease activity has been assessed by EDSS, with values ranging from 0 (normal neurological examination) to 10 (death due to MS) (
). Magnetic resonance imaging (MRI) was performed at the beginning of therapy and eventually during treatment. Finally, a lumbar puncture was performed in case of neuro–radiological or clinical alterations, concomitant to JCPyV replication in blood.
2.2 Virological investigations
JCPyV DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN, Italy). Extraction products were analyzed by a quantitative PCR (Q-PCR) system able to detect and quantify a 54 bp amplicon in JCPyV T antigen region, using a 7300 real-time PCR system (Applied Biosystems) and following published protocol (
). Each sample was analyzed in triplicate, and JCPyV DNA loads (given as the mean of at least three positive reactions) were expressed as genome equivalents (gEq)/milliliter for plasma and urine and as genome equivalents (gEq)/106 cells for PBMC. Negative and positive controls were included in each Q-PCR session. Standard curve was obtained from serial dilutions (range: 105–102 gEq/mL) of a plasmid containing the entire JCPyV genome. The lower detection limit of the Q-PCR system was 10 DNA copies of the target gene per amplification reaction, corresponding to 10 genome equivalents per reaction (10 gEq/reaction). JCPyV DNA positive samples were further analyzed using nested-PCR for NCCR and VP1 regions’ amplification (
). PCR products were analyzed on 2% agarose gels. The amplified products were purified using MinElute PCR Purification Kit (QIAGEN, Italy) and sequenced in a dedicated facility (Bio-Fab research, Italy). Obtained sequences were compared to reference strain (GenBank: AB081613). Sequence alignment was obtained using
(http://www.ebi.ac.uk/Tools/msa/clustalw2/) on the European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL–EBI) website using default parameters. JCPyV genotypes/subtypes were classified based on single nucleotide polymorphisms (SNPs) found within the amplified VP1 region (
In phylogenetic analysis, representative strains obtained from urine, plasma and PBMC were compared with JCPyV reference (AB081613) and aligned using ClustalW2. Phylogenetic tree was created using Molecular Evolutionary Genetics Analysis (Mega), using neighbor joining algorithm (
). A bootstrap test with 1000 replicates was performed to evaluate the confidence of the branching pattern of the trees.
2.4 Statistical analysis
JCPyV detection was summarized by counts and proportions. If continuous variables were normally distributed, they were expressed as mean ± SD; if not, they were expressed by median and range. The χ2 test was performed to evaluate differences in the viral detection and the Mann–Whitney U test for non-normally distributed continuous variables was applied to analyze differences between patients. A p value < 0.05 was considered statistically significant.
3. Results
3.1 Trend of JC viral load
At baseline (T0), JCPyV DNA was found in 10 out of 30 RRMS patients enrolled. Specifically, viral DNA was found in urine (viruria) of 4 patients with an average value of 3 × 104 gEq/mL and in 3 patients’ plasma (viremia), with an average value of 3.5 × 103 gEq/mL. In 2 patients, JCPyV DNA was found in PBMC with an average value of 8 × 102 gEq/106 cells. In one patient viral DNA was detected in plasma (1 × 103 gEq/mL) and in PBMC (4.5 × 102 gEq/106 cells), simultaneously (Fig. 1, Table 2). At T1 (4 natalizumab infusions), 15 RRMS patients presented JCPyV DNA: 9 had viruria (average value: 7.5 × 104 gEq/mL), 3 exhibited viremia (average value: 4 × 103 gEq/mL), whereas 2 patients showed a JCPyV DNA positivity in PBMC (average value: 1 × 103 gEq/106 cells). The only patient positive to JCPyV DNA both in plasma and PBMC at T0, was found positive at T1 yet (plasma: 9.5 × 102 gEq/mL; PBMC: 3 × 102 gEq/106 cells) (Fig. 1, Table 2). After 8 natalizumab infusions (T2), 23/30 patients presented JCPyV DNA: 15 in urine (average value: 9.5 × 104 gEq/mL), 4 were viremic (average value: 6.5 × 103 gEq/mL), 3 were positive in PBMC (average value: 1 × 103 gEq/106 cells). The simultaneous presence of JCPyV DNA found at T0 and T1 in one patient, was maintained, although with an increase in plasma (9 × 102 gEq/mL) and PBMC (3.5 × 102 gEq/106 cells) viral loads (Fig. 1, Table 2).
Fig. 1Venn diagram showing the number of patients positive to JCPyV DNA in urine (viruria), plasma (viremia) and in PBMC at different time of natalizumab infusions. The number of patients presenting JCPyV DNA in multiple samples simultaneously (plasma and PBMC, urine and plasma) (shadow zone) is also reported.
At T3 (12 natalizumab infusions), all RRMS patients revealed JCPyV DNA: 18 exhibited viruria (average value: 2.5 × 105 gEq/mL), 7 viremia (average value: 7 × 103 gEq/mL), 5 were positive in PBMC (average value: 1.5 × 103 gEq/106 cells), whereas the patient, previously positive in plasma and PBMC simultaneously, confirmed the infection only in plasma (Fig. 1, Table 2).
Results obtained after 16 natalizumab infusions (T4) showed that 22/30 patients displayed viruria (average value: 5,5 × 105 gEq/mL), 6 viremia (average value: 4 × 104 gEq/mL) and 5 were JCPyV DNA positive in PBMC (average value: 3 × 103 gEq/106 cells). The patient positive only in plasma at T3, became positive in both plasma and PBMC with an average value of 3,5 × 104 gEq/mL and 1 × 102 gEq/106 cells, respectively. Moreover, 6 patients were positive both in urine (average value: 2,5 × 105 gEq/mL) and plasma (average value: 3 × 104 gEq/mL) samples (Fig. 1, Table 2).
At T5 (20 natalizumab infusions), excretion of JCPyV DNA in urine occurred in 25 patients (average value: 9,5 × 105 gEq/mL). Nine patients presented viremia (average value: 5,5 × 104 gEq/mL), whereas 5 showed a JCPyV DNA in PBMC (average value: 4 × 103 gEq/106 cells). The patient positive to JCPyV DNA in plasma and PBMC at T0, T1, T2 and T4, showed, once again, JCPyV DNA only in plasma. The same six patients already positive in urine and plasma at T4, confirmed JCPyV DNA at T5 (urine average value: 6,5 × 105 gEq/mL; plasma average value: 3,5 × 104 gEq/mL) (Fig. 1, Table 2).
Statistical analysis of Q-PCR results indicated that the average value of JCPyV viral load detected in urine samples was significantly higher than that observed in plasma and in PBMCs (p ≤ 0.05) (Fig. 2).
Fig. 2Statistical analysis of the JC viral load with respect natalizumab infusions in urine, plasma and PBMC during the whole study. The average value of JC viruria was always significantly higher than JC viremia average value. Statistical analysis was performed using nonparametric tests and significance was set for a p value <0.05.
3.2 Contextual analysis of the anti-JCPyV antibodies versus JCPyV-DNA detection
Before starting natalizumab treatment (T0) and after 12 natalizumab infusion (T3), a second-generation assay (STRATIFY JCVⓇ DxSelect™), was performed on serum of the 30 RRMS subjects. At T0, JCPyV-specific antibodies were observed in serum of 8 patients (STRATIFY JCVⓇ-positive), whereas 22 showed a negative STRATIFY JCV® (Table 3). Contextually to the analysis of the anti-JCPyV antibodies, 10/30 patients revealed a JCPyV-DNA positivity: 1 among 8 STRATIFY JCVⓇ-positive patients and 9 among STRATIFY JCV®-negative patients. In particular, the STRATIFY JCVⓇ-positive patient presented viral DNA in plasma and in PBMC, simultaneously (Table 3). By contrast, among 9 STRATIFY JCVⓇ-negative patients, 4 exhibited JC viruria, 3 were viremic and 2 showed a JCPyV DNA in PBMC (Table 3). At T3, the scenario changed: the number of STRATIFY JCVⓇ-positive patients increased from 8 to 12, whereas all RRMS patients revealed JCPyV DNA.
Table 3Contextual analysis of the anti-JCPyV antibodies versus JCPyV-DNA detection. At T0, JCPyV-specific antibodies were observed in serum of 8 patients whereas 22 showed a negative STRATIFY JCVⓇ. Contextually to the analysis of the anti-JCPyV antibodies, JCPyV-DNA detection showed a positivity in 10/30 RRMS patients: 1 among 8 STRATIFY JCVⓇ-positive patients and 9 among STRATIFY JCVⓇ-negative patients. Only one STRATIFY JCVⓇ-positive patient presented viral DNA in plasma and in PBMC, simultaneously. Among 9 STRATIFY JCVⓇ-negative patients, 4 had JC viruria, 3 were viremic and 2 were in PBMC. At T3 the number of STRATIFY JCVⓇ-positive patients was 12, whereas all RRMS patients revealed JCPyV DNA. Among 12 STRATIFY JCVⓇ-positive patients, 5 had viruria, 4 viremia and 3 were positive in PBMC. In the remaining 18 STRATIFY JCVⓇ-negative patients, 13 resulted JCPyV DNA positive in urine, 3 were positive in plasma and 2 were positive in PBMC.
T0: baseline
Patients stratify JCVⓇ +
Patients JCPyV DNA +
Contextual positivity to stratify JCVⓇ and JCPyV DNA
SAMPLE OF JCPyV-DNA DETECTION
8/30
10/30
1/8
Plasma and PBMC (1)
Patients stratify JCVⓇ -
22/30
10/30
9/22
Urine (4), Plasma (3), PBMC (2)
T3: 12 Natalizumab infusions
Patients stratify JCVⓇ +
Patients JCPyV DNA +
Contextual positivity to stratify JCVⓇ and JCPyV DNA
Specifically, among 12 STRATIFY JCVⓇ-positive patients, 5 (STRATIFY JCVⓇ-negative and negative for JCPyV viruria at T0) had viruria and 4 viremia (1 already STRATIFY JCVⓇ-positive and viremic at T0 and the remaining 3 featured a new diagnosis of viremia) (Table 3). Finally, 3/12 T3-STRATIFY JCVⓇ-positive patients were positive for JCPyV DNA in PBMC: 1 patient, at T0, was STRATIFY JCVⓇ-positive and positive for JCPyV DNA in PBMC, whereas, the residual 2 patients presented a new positivity for JCPyV in PBMC, at T3 (Table 3). Among the remaining 18 STRATIFY JCVⓇ-negative patients, JCPyV-DNA detection provided positive results in all analyzed patients. Specifically, 13 patients resulted JCPyV DNA positive in urine (the number increased from 4 to 13), whereas patients, positive to DNA detection in plasma (3 samples) and in PBMC (2 samples), conserved the same positivity, previously diagnosed at T0 (Table 3).
3.3 NCCR and VP1 sequences’ evaluation
At T0, JCPyV NCCR analysis showed a structural organization similar to archetype JCPyV in all RRMS patients’ samples (Fig. 3A). During the follow-up less than 12 months, point mutations were identified. In particular, in plasma of a patient T0-T3 STRATIFY JCVⓇ-negative after one year of natalizumab treatment, a nucleotide transition 208A→G was found inside box F, involving NF-1 binding site (Fig. 3B). In plasma and PBMC samples of two different patients T0-T3 STRATIFY JCVⓇ-negative, NCCR sequence showed a 37T→G transversion in box B, inside the binding site for the cellular transcription factor Spi-B (Fig. 3C). Finally, a sequence obtained from plasma and PBMC, belonging to the only patient T0-T3 STRATIFY JCVⓇ-positive, displayed the transition 217G→A in box F (Fig. 3D). Analysis of NCCR of RRMS patients, treated with natalizumab for more than 12 months, showed identical point mutations already observed in plasma and PBMC during the first year of natalizumab infusions, in the same patients. Remarkably, plasma of T0-T3 STRATIFY JCVⓇ-negative patient, that displayed the transversion 37T→G in Spi-B binding site (box B), showed, after 16 natalizumab infusions (T4), an arranged NCCR, characterized by a duplication of NF-1 cellular transcription factor binding site (box F) and by a box D composed only of 10 out of 65 bases (nucleotides from 117 to 126). The resulted NCCR was A– B*–C–(D)–E–F* (Fig. 4A). Analysis of PBMC, belonging to a patient T0-T3 STRATIFY JCV®-negative and with a transversion 37T→G in Spi-B binding site (box B), displayed, after 20 natalizumab infusions (T5), an arranged NCCR with duplication of box C and deletion of nucleotides from 117 to 180, corresponding to box D. This deletion conferred the following NCCR-box organization: A–B*–C–C–E–F (Fig. 4B).
Fig. 3NCCR analysis during the firt year of natalizumab treatnment (T0–T3). A: an archetype JCPyV NCCR, observed in all RRMS patients’ samples at T0, was showed. B: after one year of natalizumab infusions, a nucleotide transition 208A→G was found inside box F (involving NF–1 binding site) in plasma of a patient T0–T3 STRATIFY JCVⓇ-negative. C: NCCR sequence showed a 37T→G transversion in box B (inside Spi-B binding site) in plasma and PBMC samples of two different patients, T0–T3 STRATIFY JCV®-negative. 3D: NCCR obtained from plasma and PBMC, belonging to the only patient T0–T3 STRATIFY JV®-positive, displayed the transition 217G→A in box F.
A: at T4, plasma of T0–T3 STRATIFY JCVⓇ-negative patient displayed NCCR with the transversion 37T→G in Spi-B binding site (box B), duplication of NF-1 cellular transcription factor binding site (box F) and a box D composed only of 10 out of 65 bases (nucleotides from 117 to 126). The resulted NCCR was A– B*–C–(D)–E–F*.
B: at T5, PBMC, belonging to a patient T0-T3 STRATIFY JCV®-negative, showed a transversion 37T→G in Spi-B binding site (box B), duplication of box C and deletion of nucleotides from 117 to 180, corresponding to box D. This deletion conferred the following NCCR-box organization: A–B*–C–C–E–F.
Regarding JCPyV VP1 sequence analysis, results showed that genotypes 1A and 1B were always observed for the whole duration of the study. After 12 months of natalizumab infusions, in two different patients STRATIFY JCVⓇ-negative at T3, the amino acid change mutations S269F in plasma and S267L in PBMC, involving the receptor-binding region of VP1, were observed. In detail, S267L corresponded to 1A genotype whereas S269F was related to 2B genotype.
3.4 Phylogenetic analysis
Phylogenetic analysis, carried out on VP1 sequences, revealed that all isolates were 99% identical to the reference AB081613, suggesting a stability and a similarity across different isolates of JCPyV VP1.
4. Discussion
Since PML is the main restriction to natalizumab administration, it is crucial to stem this limitation by identifying biomarkers able to support the established factors for PML risk stratification on single RRMS patients.
To achieve this goal, our study was addressed to assess if JCPyV-DNA detection could play a role of viral reactivation and infection's biological marker. Our results showed that JC viral DNA was detected in urine, plasma and PBMC for the whole study, although JC viral load in urine was significantly higher compared to viremia at all selected time points. This result could be explained assuming that, the decrease of immunosurveillance at the urinary level, caused by natalizumab, determines, firstly, a higher JCPyV replication in renal cells with a viral shedding and, as expected from JCPyV biology, a subsequent release of JCPyV–infected cells into bloodstream (
). Moreover, reduced immunosurveillance determined an increment in JC viral load, from T0 to T5, which followed a linear trend with natalizumab infusions’ number (
). To further corroborate that viral DNA detection could be considered a valid JCPyV alert, a contextual analysis of anti-JCPyV antibodies versus JC viral load, was carried out at T0 and at T3. Results of STRATIFY JCVⓇ did not always correlate with Q-PCR data. In fact, at baseline, only 1 patient exhibited anti-JCPyV antibodies and viral DNA in plasma and in PBMC, simultaneously. At T3, although the number of STRATIFY JCVⓇ-positive patients increased to 12, each RRMS patients presented JCPyV DNA, probably ascribed to JCPyV reactivation's occurrence at visit calendar time, when anti-JCPyV antibodies are still absent, despite viral replication, and to a low anti-JCPyV antibody titer, not detectable by STRATIFY JCVⓇ assay (
). In fact, although second-generation STRATIFY JCVⓇ showed an increased sensitivity, some patients present a negativity to anti-JCPyV antibodies and a detectable JCPyV DNA in urine or blood. Our data confirm that anti-JCPyV antibodies assay, alone, is not sufficient for JCPyV diagnosis and should be associated with JCPyV-DNA detection in different body compartments, in order to accurately identify JCPyV infected patients (
). Consequently, different approaches to detect JCPyV infection are required for a more accurate PML risk monitoring and for the evaluation of those patients, who should start or are already receiving natalizumab. A restriction to JCPyV-DNA detection, as a predictive PML-biomarker, could be that JCPyV specific Q-PCR is not able to discriminate between archetype and rr-NCCR. Development of a Q-PCR assay, that specifically identify neurotropic strains, could be an important advance in PML diagnostics (
Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants.
), although it is more difficult, owing to the multitude of arrangements that can occur to yield a more virulent virus. In this framework, since it is possible to speculate that the higher the JC viremia, the higher the possibility that the pathogenic JCPyV emerges, consider Q-PCR data and consequently analyze NCCR sequence could be useful to identify variants correlated with PML. Sequence analysis exhibited an archetype-like structural organization in all analyzed samples during the follow-up less than 12 months, although point mutations, previously observed by other authors in vivo and in vitro (
Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.
), were identified in plasma of 3 T0–T3 STRATIFY JVⓇ-negative patients and in plasma and PBMC of 1 T0-T3 STRATIFY JVⓇ-positive patient.
It is important to remark that the 37T→G transversion in Spi-B binding site of box B, preceded NCCR re-organization and, in particular, it is able to transform archetype Spi-B binding site in a JCPyV PML-variant. This change could enhance JCPyV replication, especially in glial cells, B cells and hematopoietic progenitor cells, in which Spi-B has been shown to be up-regulated (
In one plasma's patient, in addition to transversion 37T→G, JCPyV NCCR showed a rearranged structure with duplication, involving NF-1 binding site (box F), and a box D composed of nucleotides from 117 to 126. It is known that NF-1 increased the expression of JCPyV's early and late genes, promoting onset of JCPyV variants with determinants of neurotropism and increased neurovirulence (
). Instead, in PBMC of a patient with 20 natalizumab infusions (T5), in addition to 37T→G mutation, JCPyV-NCCR showed an organization of type A–B*–C–C–E–F, with a duplication of box C, which includes duplication of CRE element binding site, and deletion of box D. The potential neurovirulence of this sequence was conferred by repetition of CRE element, a specific enhancer of JCPyV replication in glial cells in box C (
Glial and muscle embryonal carcinoma cell-specific independent regulation of expression of human JC virus early promoter by cyclic amp response elements and adjacent nuclear factor 1 binding sites.
). NCCR box duplications were recurrently found near to the origin of genome replication (left box A to C), whereas deletions were more frequent near to late genes (right box D to F) (
). Duplications in the left end of NCCR represent a gain of function, increasing activating control sequences that enhance viral replication and gene transcription whereas deletions, in proximity of late genes, determine a loss of function, removing a suppressing control sequence (
Regarding VP1 analysis, although this gene is highly polymorphic, only in two different T3 STRATIFY JCVⓇ-negative patients, mutations S269F and S267L were observed in plasma and in PBMC, respectively. Therefore, variations in VP1 region could change the virus's binding properties to its receptor or drive to alternative receptor usage (
Although JCPyV NCCR arrangements and VP1 mutations are independent events that impact distinct elements of JCPyV cellular tropism, our results highlight to consider NCCR and VP1 modifications as biomarkers to monitor the onset of neurovirulent variants.
5. Conclusion
To conclude, we highly recommend considering JCPyV-DNA detection in order to accurately identify JCPyV infected patients with a specific antibodies not yet detectable. Moreover, since a higher JC viremia linked to the possibility that neuro-pathogenic JCPyV emerges, monitoring the trend of JC viral load could be a useful tool to identify mutations and/or NCCR re-organization correlated with neuro-virulent variants. Taking in account these viral biomarkers could improve PML risk stratification supporting a timely diagnosis, optimizing health resources and contributing to the reduction of direct and indirect costs of MS disease.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Funding
Anna Teresa Palamara: Funding acquisition (MIUR-PRIN Grant: 2015W729WH); Valeria Pietropaolo: Funding acquisition (MIUR Research Grant: RP118163D5757CA6).
CRediT authorship contribution statement
Carla Prezioso: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Maria Antonella Zingaropoli: Conceptualization, Methodology, Writing - review & editing, Data curation. Marco Iannetta: Conceptualization, Methodology, Writing - review & editing. Donatella Maria Rodio: Conceptualization, Methodology, Writing - review & editing. Marta Altieri: Resources, Data curation. Antonella Conte: Resources, Data curation. Vincenzo Vullo: Writing - review & editing. Maria Rosa Ciardi: . Anna Teresa Palamara: Funding acquisition, Writing - review & editing. Valeria Pietropaolo: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgments
Carla Prezioso was supported by funding dispensed by Italian Ministry of Health (starting Grant: SG-2018-12366194).
References
Berger J.R.
Classifying PML risk with disease modifying therapies.
Glial and muscle embryonal carcinoma cell-specific independent regulation of expression of human JC virus early promoter by cyclic amp response elements and adjacent nuclear factor 1 binding sites.
Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.
Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants.
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