Human Polyomavirus JC monitoring and noncoding control region analysis in dynamic cohorts of individuals affected by immune-mediated diseases under treatment with biologics: an observational study

Abstract

Background: Progressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by immune-mediated diseases during biological treatment, raised concerns about the safety profile of these agents. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by different immune-mediated diseases and treated with biologics.

Methods: We performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3, t4). Subsequently, rearrangements' analysis of NCCR and VP1 was carried out. Data were analyzed using χ2 test.

Results: Results showed that at t0 patients with chronic inflammatory rheumatic diseases presented a JCPyV load in the urine significantly higher (p≤0.05) than in patients with multiple sclerosis (MS) and Crohn's disease (CD). It can also be observed a significant association between JC viruria and JCPyV antibodies after 1 year of natalizumab (p=0.04) in MS patients. Finally, NCCR analysis showed the presence of an archetype-like sequence in all urine samples, whereas a rearranged NCCR Type IR was found in colon-rectal biopsies collected from 2 CD patients after 16 months of infliximab. Furthermore, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 MS patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98 bp unit and a 66 bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed.

Conclusions: It has been important to understand whether the specific inflammatory scenario in different immune-mediated diseases could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement.

Figure 1

Comparison of baseline (t0) JC viruria in 21 MS, 18 CIRDs and 22 CD patients. JCPyV DNA [(median log10 JCPyV load (range)] was found in 2/21 multiple sclerosis patients [3.90 gEq/mL (3.70-4.10)], in 14/22 CIRDs patients [7.21 gEq/mL (4.15-8.13)] and in 6/18 CD patients [6.30 gEq/mL (4.85-8.85)]. Moreover 5/19 healthy individuals showed JCPyV DNA in urine [5.75 gEq/mL (3.54-8.43)]. Comparing these data of JC viruria, we observed that CIRDs patients presented a JC viruria significantly higher than that presented by patients with MS or with CD and by healthy individuals. JCPyV loads values are expressed as log10 genome equivalent (gEq)/mL. Comparisons were performed using non parametric Mann–Whitney U-test and statistically significant p values (< 0.05) were indicated.

Figure 2

Comparison of JC viremia and viruria in 1-years follow-up: 21 MS vs 18 CD patients. (A.) Comparing JC viremia follow up of MS vs CD patients, the viremia of CD patients during the follow-up is numerically higher than that observed in MS patients, although we did not find a statistically significant difference (p = 0.07). (B.) Comparing JC viruria follow up of MS patients treated with natalizumab vs CD patients treated with infliximab, we observed a persistent viruria both cohorts. However the JC viruria of patients treated with infliximab, was significantly higher than that of patients treated with natalizumab only at t1 (p = 0.01) and at t3 (p = 0.05). Moreover a significant difference between the JC viruria detected at t0 and at t3 in both cohorts was also reported. JCPyV load values are expressed as log10 genome equivalent (gEq)/mL. JCPyV load values of MS cohort and CD cohort are reported in table 1 and table 4, respectively. Comparisons were performed using non parametric Mann–Whitney U-test and p values <0.05 were considered statistically significant. t0: baseline values. t1, t2 and t3: 4, 8 and 12 months of therapy, respectively.

Figure 3

JCPyV NCCR sequence analysis in 21 MS and 18 CD patients. (A.) Type IR has repeats of 98bp unit, also known as sequence composed by boxA (25 bp), boxC (55 bp) and boxE (18 bp), as seen in the JCPyV prototype Mad-1 [35,36], without 23- and/or 66-bp inserts, also known as boxB and boxD respectively. (B.) Type IIS has a single 98 bp unit, with one 23- and one 66-bp insert, as seen in the archetype CY [35,38]. The NCCR type IIS is also known as sequence composed by boxes A, B, C, D and E. Each 98bp unit is represented by an open box. The 23 bp and 66 bp inserts are represented as open boxes labeled “23 bp” and “66 bp,” respectively. The nucleotide numbering of Mad-1 NCCR is indicated in black bold font, whereas the nucleotide numbering of CY NCCR is reported in grey bold font. In all NCCR sequences is present a 69 bp sequence, called box F, starting from nucleotide 207. (C.) A NCCR Type IIS was found in 8/28 plasma, 7/21 ileal and 5/16 colon-rectal biopsies, collected from CD patients. A NCCR Type IIR with a boxD deletion was found in 4/21 ileal and 1/16 colon-rectal biopsies. Another NCCR Type IIR, composed by a duplication of a 98 bp unit and a 66 bp insert, was found in 2/3 PBMCs of 2 MS patients. Finally, a NCCR Type IR sequence was found in 2/16 colon-rectal biopsies. Dotted lines represent deletions or regions not present. Asterisks represent single nucleotide point mutations or deletions. Black bold letters indicate nucleotides and underlined letters in grey bold font indicate the relative point mutations. The types of NCCR are indicated. The main cellular factor binding sites are also reported. Ori: replication’s origin.