Natalizumab y virus jc

Try out PMC Labs and tell us what you think. Learn More. Under these experimental conditions, with respect to the observed frequency of JCPyV infection, the sensitivity of the anti-JCPyV antibody assay was lower than expected. Based on this assumption, an assay that is highly sensitive and specific for the detection of anti-JCPyV Abs in serum Stratify was developed. Paired blood and urine samples from 39 patients who were consecutively referred to the outpatient clinic of the Regional Reference Center for Multiple Sclerosis of the Careggi University Hospital, Florence, Italy, were prospectively collected.

The patients were recruited according to the following inclusion criteria: definite diagnosis of MS; relapsing-remitting course; fulfillment of the European Medicines Agency indications for natalizumab administration in MS; and written informed consent to participate in a program for PML risk stratification established in collaboration with the Marketing Authorization Holder MAH of natalizumab and based on a centralized anti-JCPyV Ab assay in sera and on a locally organized JCPyV DNA analysis in blood and urine.

The results included in the present study were generated by analyzing the diagnostic data reported in the clinical records. OSS Each assay was carried out with negative controls no template and DNA fold dilutions 10 1 —10 6 copies of a plasmid carrying the amplicon cloned standard curve plot was 10 1 —10 6 copies vs cycle threshold.

The blood and urine samples were collected biannually at baseline and during the therapy, from July to September Patients received between 0 and 35 infusions and contributed between 1 and 8 samples. Eleven out of 39 samples included were collected at baseline, and the other 28 were collected during natalizumab administration. No obvious associations between patient demographic and clinical characteristics age, sex, disease duration, previous treatments and JCPyV DNA status were observed.

The range of viral load was similar in the different compartments table 2. Baseline demographic and clinical characteristics of the patients according to JCPyV status. However, in this context, the viral load observed in seronegative patients did not differ from that observed in the seropositive patients. The previous data have been obtained by testing the virus DNA only in urine, whereas more recent data and the present study indicate that when peripheral blood is also tested the frequency of JCPyV DNA is higher than previously described, particularly during natalizumab treatment.

This possibility points out the need to reevaluate the PML risk thus far reported in anti-JCPyV seronegative patients through additional virologic investigations. Supplemental data at Neurology. Clausi contributed to data collection, data analysis, and interpretation of the data. Giannecchini contributed to data analysis, interpretation of the data, drafting of the manuscript, and revision of the manuscript.

Magnani contributed to patient recruitment and follow-up and interpretation of the data. Repice contributed to patient recruitment and follow-up and collection, analysis, and interpretation of the data. Mechi contributed to patient recruitment and follow-up and collection, analysis, and interpretation of the data. The increased frequency of T cells that produce IL after one dose of natalizumab may indicate its potential to skew the functional profiles of T cells. P value from month 0 to month 1 is the result of Wilcoxon matched-pairs rank sum test.

One of these patients, PML-4, was one of the first cases diagnosed in , who was followed for more than 5 years and never cleared JCV from the brain [29]. The observation that the frequency of IL producing memory CD4 T cells was high in subjects with natalizumab-associated PML raises the intriguing possibility that the transient increase in these cells after treatment initiation might indicate a potential role for natalizumab in skewing the immune response toward a cytokine associated with PML.

Approximately 2. We hypothesized that such individuals might have an aberrant T cell response to JCV. Given our findings that JCV-specific T cells may target many of the JCV antigens and are varied in functional profile among individuals, we sought to identify whether there were differences in the antiviral immune responses of individuals who developed PML after treatment with natalizumab.

These were the only subjects in whom no JCV-specific T cell responses were observed, as all the MS patients and healthy volunteers had detectable responses. Both subjects were still being followed 2 years after diagnosis.

Panel A shows the total response to JCV. Subjects PML-1, 2, 3 and 4 were sampled 2 weeks, 2 months, 4 months and 5 years, respectively, after diagnosis with PML. The specificity of the IL response for PML-4 was confirmed by double staining with the same antibody conjugated to two different fluorophores Figure S2. The antigen specificity of the subjects' responses differed.

In subject PML-3, 1. This subject died 1 month after sampling. In subject PML-4, 0. Subject PML-4 died 1 year after sampling. Importantly, these were the only ILproducing virus-specific T cell responses we observed among all the subjects examined in this study.

As JCV-specific T cells in the blood of the four individuals with PML were either absent or of unusual functionality, we next examined the cytokine profile within CSF samples from 10 individuals with natalizumab-associated PML, including subjects PML 1—4, at the time of initial diagnosis.

In addition, a second CSF sample from a later time point was available for 8 of these subjects. The samples were tested for the presence of 27 cytokines and other markers and were compared to diagnostic CSF samples from 10 individuals who had suspected natalizumab-associated PML which was subsequently ruled out by a negative PCR for JCV. Although the assay was not sensitive enough for accurate quantification of low levels of IL, it could readily distinguish whether IL was detectable above background.

Panel A shows the proportion of CSF samples with detectable IL in diagnostic samples from subjects in whom natalizumab-associated PML was confirmed or ruled out, and the proportion of CSF samples in each group with IL-5 levels above the lower limit of quantification 1.

P values were calculated by Fisher's exact test. P values between paired longitudinal samples were calculated by Wilcoxon matched-pairs signed rank test. The factors that lead to the development of PML in individuals treated with natalizumab need to be investigated in more detail, particularly immune responses to the virus.

Consequently, we investigated the T cell immune response to the entire JC virus proteome longitudinally in subjects with MS who were initiating therapy with natalizumab and in subjects who had natalizumab-associated PML. Whether JCV-specific T cell responses can be reliably measured ex vivo [30] , [31] and the effect of natalizumab on these responses [23] , [24] have both been the subject of some debate.

This approach could be useful in monitoring JCV-specific T cell function in natalizumab-treated individuals in the context of a vaccine or a risk stratification protocol. Our findings that JCV-specific T cells are directed against each of the viral proteins and that the specificity and immunodominance of the response varies among individuals strongly suggest that measuring responses to all viral proteins rather than VP1 alone is essential to obtaining a complete picture of JCV-specific immunity.

In our longitudinal study we found no difference in either the magnitude or functional profile of the total JCV-specific T cell response during short-term natalizumab treatment. Although the sample size of our study was limited in power to detect longitudinal differences, the fact that no difference was observed in any of the cytokines measured, or in their relative contribution to the response at different time points, suggests that JCV-specific T cell responses are not altered simply by the initiation of natalizumab.

Importantly, it has been shown that IL is detrimental to the clearance of lymphocytic choriomeningitis virus LCMV infection because of its inhibitory effect on virus-specific memory CD4 T cells [25] , [26]. This sample size does not allow us definitively to link the time since diagnosis to the presence of an IL response.

Importantly, these two interpretations are not mutually exclusive. This result suggests that IL may be associated with PML resulting from causes other than natalizumab therapy. Notably, we found that the frequency of memory CD4 T cells that produce IL upon mitogenic stimulation is transiently increased after the first dose of natalizumab.

There is also the possibility that a direct effect of natalizumab on T cells could affect cytokine production, as may occur with ribavirin treatment for hepatitis C virus [33]. Furthermore, in this study we were only able to measure T cell responses in the peripheral blood and were not able to sample the CNS of subjects without PML.

Although this finding may be due to altered CSF cell subset composition after treatment rather than upregulation of IL, it supports the hypothesis that natalizumab may alter the cytokine milieu in the CNS.

Although these cytokines are not typically associated with control of virus replication or with each other, their aberrant production may be indicative of an immune response that fails to control JCV replication at the site of disease. Thus far, it is not possible to demonstrate a causative link between IL production and PML as the very low incidence of natalizumab-associated PML makes a prospective study unlikely.

However, we believe that the potential mechanism suggested by these data should inform future work. Taken together, our data provide a framework for understanding immune control of JC viremia and the development of PML and suggest avenues of investigation to allow the better monitoring, treatment and prevention of PML in natalizumab-treated people.

First, our finding that subjects with PML lacked JCV-specific T cell responses or produced IL in response to stimulation suggests that immune monitoring might identify natalizumab-treated individuals who are at risk of developing PML, by screening subjects prior to treatment or while on treatment.

None of the MS patients without PML or healthy subjects included in our study showed an absent or IL producing T cell response similar to that observed in the subjects with PML, and this suggests that individuals with these phenotypes are relatively rare and could be identified by immune monitoring prior to treatment. Written informed consent was obtained from all study subjects. Subjects underwent a washout period of at least two weeks and had blood samples taken immediately before the first dose and each subsequent monthly infusion.

Tables S1 , S2 , S3 describe certain clinical and laboratory characteristics of all the included natalizumab-treated patients. Cells were then rested for two hours before being washed and then plated in well plates in uL final volume of R10 with DNase I.

Flow cytometric analysis was done within 24 h of staining. Between , and 1,, events were collected for each sample. Electronic compensation was conducted with antibody capture beads BD Biosciences stained separately with individual mAbs used in test samples. All analytical gating was performed using FlowJo version 9.

Ashland, OR. CD4 and CD8 memory T cells were identified by sequential gating, using the same gating scheme for all analyzed samples. An amino acid sequence for the entire JCV coding region was constructed based on Mad1 that also included any common variants from NCBI and the literature [40] , in order to cover the vast majority of variation in the viral epitopes that are presented in vivo in our patient population particularly focusing on genotypes 1, 3, 4 and 6.

Because the peptide stimulations were done in pools rather than individually, and contained multiple variants of the same peptide, it is theoretically possible that competition amongst peptides in the same pool for the same MHC protein decreased sensitivity of the assay to detect a response. Limitations on the number of cells available from samples prevents the assessment of all peptides individually and necessitates the commonly-used pooling approach. Experimental variables were analyzed using Fisher's Exact test, Mann-Whitney U test or Wilcoxon matched-pairs signed rank test.

Bars depict median values. JCV-specific memory T cell responses in healthy subjects. CD4 top and CD8 bottom memory T cell responses from all ten healthy subjects are shown, with the background-subtracted magnitude of the response to each JCV protein depicted by colored bars. It is possible that such responses may be specific for BK virus, as it shares significant homology with JCV.

As negative serology does not definitively rule out latent JCV infection one cannot fully distinguish JCV-infected from uninfected subjects cross-sectionally. However, the presence of T cells that respond to JCV, whether or not these cells were originally primed to JCV itself, has implications for the control of new infection or reactivation of latent JCV.

Methods: We followed 19 consecutive patients with multiple sclerosis who were treated with natalizumab over an month period, performing quantitative polymerase-chain-reaction assays in blood and urine for JC virus reactivation; BK virus, a JC virus-related polyomavirus, was used as a control.

We determined JC virus-specific T-cell responses by means of an enzyme-linked immunospot assay and antibody responses by means of an enzyme-linked immunosorbent assay and analyzed JC virus regulatory-region sequences. JC virus regulatory-region sequences in blood samples and in most of the urine samples were similar to those usually found in PML.