Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy

JC virus (JCV)-specific cytotoxic T lymphocytes (CTL) in peripheral blood are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML). However, the frequency of these cells in the peripheral blood mononuclear cells (PBMC) of PML patients is unknown. To develop a highly sensitive assay for detecting the cellular immune response against this virus, we performed a CTL epitope mapping study of JCV VP1 major capsid protein by using overlapping peptides. A novel HLA-A*0201-restricted epitope, the VP1(p36) peptide SITEVECFL, was characterized. The cellular immune response against JCV was assessed in 32 study subjects. By combining the results of the (51)Cr release assay on pooled peptides and staining with the HLA-A*0201/JCV VP1(p36) tetramer, VP1-specific CTL were detected in 10 of 11 PML survivors (91%) versus only 1 of 11 PML progressors (9%, P = 0.0003). VP1-specific CTL were also detected in two of two patients recently diagnosed with PML and in four of four human immunodeficiency virus-positive patients with possible PML. The frequency of CTL specific for the novel VP1(p36) and the previously described VP1(p100) epitopes was determined. In two patients, the frequency of CTL specific for the VP1(p36) or VP1(p100) epitopes, as determined by fresh blood tetramer staining (FBTS), ranged from 1/6,000 to 1/24,000 PBMC. A CTL sorting technique combining tetramer staining and selection with immunomagnetic beads allowed the detection of epitope-specific CTL in two cases that were determined to be negative by FBTS. The phenotype of these CTL in vivo was consistent with activated memory cells. These data suggest that, although present in low numbers, JCV-specific CTL may be of central importance in the containment of JCV spread in immunosuppressed individuals.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Detection of JC virus-specific cytotoxic T lymphocytes in healthy individuals

The polyomavirus JC (JCV) infects 85% of healthy individuals, and its reactivation in a limited number of immunosuppressed people causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system. We hypothesized that JCV-specific cytotoxic T lymphocytes (CTLs) might control JCV replication in healthy individuals, blocking the evolution of PML. Using 51Cr release and tetramer staining assays, we show that 8 of 11 HLA-A*0201+ healthy subjects (73%) harbor detectable JCV-specific CD8+ CTLs that recognize one or two epitopes of JCV VP1 protein, the HLA-A*0201-restricted VP1p36 and VPp1100 epitopes. We determined that the frequency of JCV VP1 epitope-specific CTLs varied from less than 1/100,000 to 1/2,494 peripheral blood mononuclear cells. More individuals had JCV VP1-specific than cytomegalovirus-specific CTLs (8 of 11 subjects [73%] versus 2 of 10 subjects [20%], respectively). These results show that a CD8+-T-cell response against JCV is commonly found in immunocompetent people and suggest that these cells might protect against the development of PML.     

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.     

Changes in JC Virus-Specific T Cell Responses during Natalizumab Treatment and in Natalizumab-Associated Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients.     

Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy

Reactivation of the polyomavirus BK (BKV) causes polyomavirus nephropathy (PVN) in kidney transplant (KTx) recipients and may lead to loss of the renal allograft. We have identified two HLA-A*0201-restricted nine-amino-acid cytotoxic T lymphocyte (CTL) epitopes of the BKV major capsid protein VP1, VP1(p44), and VP1(p108). Using tetramer staining assays, we showed that these epitopes were recognized by CTLs in 8 of 10 (VP1(p44)) and 5 of 10 (VP1(p108)) HLA-A*0201+ healthy individuals, while both epitopes elicited a CTL response in 10 of 10 KTx recipients with biopsy-proven PVN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1(p44) were more abundant than against VP1(p108) in most healthy individuals, while the converse was true in KTx recipients with PVN, suggesting a shift in epitope immunodominance in the setting of active BKV infection. A strong CTL response in KTx recipients with PVN appeared to be associated with decreased BK viral load in blood and urine and low anti-BKV antibody titers, while a low or undetectable CTL response correlated with viral persistence and high anti-BKV antibody titers. These results suggest that this cellular immune response is present in most BKV-seropositive healthy individuals and plays an important role in the containment of BKV in KTx recipients with PVN. Interestingly, the BKV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus JC (JCV), JCV VP1(p36) and VP1(p100). A high degree of epitope cross-recognition was present between BKV and corresponding JCV-specific CTLs, which indicates that the same population of cells is functionally effective against these two closely related viruses.     

Improved survival of HIV-1-infected patients with progressive multifocal leukoencephalopathy receiving early 5-drug combination antiretroviral therapy

Background

Progressive multifocal leukoencephalopathy (PML), a rare devastating demyelinating disease caused by the polyomavirus JC (JCV), occurs in severely immunocompromised patients, most of whom have advanced-stage HIV infection. Despite combination antiretroviral therapy (cART), 50% of patients die within 6 months of PML onset. We conducted a multicenter, open-label pilot trial evaluating the survival benefit of a five-drug cART designed to accelerate HIV replication decay and JCV-specific immune recovery.

Methods and Findings

All the patients received an optimized cART with three or more drugs for 12 months, plus the fusion inhibitor enfuvirtide during the first 6 months. The main endpoint was the one-year survival rate. A total of 28 patients were enrolled. At entry, median CD4+ T-cell count was 53 per microliter and 86% of patients had detectable plasma HIV RNA and CSF JCV DNA levels. Seven patients died, all before month 4. The one-year survival estimate was 0.75 (95% confidence interval, 0.61 to 0.93). At month 6, JCV DNA was undetectable in the CSF of 81% of survivors. At month 12, 81% of patients had undetectable plasma HIV RNA, and the median CD4+ T-cell increment was 105 per microliter. In univariate analysis, higher total and naive CD4+ T-cell counts and lower CSF JCV DNA level at baseline were associated with better survival. JCV-specific functional memory CD4+ T-cell responses, based on a proliferation assay, were detected in 4% of patients at baseline and 43% at M12 (P = 0.008).

Conclusions

The early use of five-drug cART after PML diagnosis appears to improve survival. This is associated with recovery of anti-JCV T-cell responses and JCV clearance from CSF. A low CD4+ T-cell count (particularly naive subset) and high JCV DNA copies in CSF at PML diagnosis appear to be risk factors for death.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00120367","term_id":"NCT00120367"}}NCT00120367     

JC virus encephalopathy is associated with a novel agnoprotein-deletion JCV variant

JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCV(CPN1), with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti- VP1 antibodies demonstrated that the truncated JCV(CPN1) Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+ and HIV- control subjects. Another JCV(CPN) strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wild-type, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCV(CPN1), may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild-type strain of JCV.     

Cross-reactive cytotoxic T lymphocytes against human immunodeficiency virus type 1 protease and gamma interferon-inducible protein 30

The gamma interferon (IFN-gamma)-inducible protein 30 (IP-30) signal peptide -11 to -3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-gamma, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-gamma. Neither high levels of exogenous IFN-gamma nor incubation of PBMC with other HIV peptides triggering substantial IFN-gamma release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-gamma release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-gamma release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide -11 to -3, which has implications for immune memory and autoimmunity.     

T Cell Epitope Mapping of JC Polyoma Virus-Encoded Proteome Reveals Reduced T Cell Responses in HLA-DRB1*04:01+ Donors

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4⁺ T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4⁺ T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01⁺, DRB1*07⁺, DRB1*11⁺, DRB1*13⁺, DRB1*15⁺, and DRB1*03⁺ individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01⁺ individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.     

Role of CD4+ and CD8+ T-cell responses against JC virus in the outcome of patients with progressive multifocal leukoencephalopathy (PML) and PML with immune reconstitution inflammatory syndrome

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the brain caused by JC virus (JCV). To assess the role of CD4(+) and CD8(+) T-cells against JCV in the clinical outcome of PML and PML in the setting of immune reconstitution inflammatory syndrome (IRIS), we tested gamma interferon (IFN-γ) response by enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) in 117 subjects, including 66 PML patients with different clinical outcomes. Both assays were concordant and demonstrated that the cellular immune response against JCV is associated with better clinical outcome. PML survivors had an early CD8(+) T-cell response more frequently than PML progressors (100% versus 27.3%; P = 0.001), while only a trend was observed for the early CD4(+) T-cell response between these two groups (80% versus 45.5%; P = 0.18). Although IRIS itself was more frequent in the PML survivor group, there was no difference in IFN-γ-producing CD4(+) and CD8(+) T-cells between IRIS and non-IRIS PML patients, suggesting that T-cells expressing other cytokines likely have a role in the immunopathogenesis of IRIS. ELISpot and ICS assays are useful prognostic markers of PML evolution and may help in the clinical management of these patients.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

JC virus regulatory region tandem repeats in plasma and central nervous system isolates correlate with poor clinical outcome in patients with progressive multifocal leukoencephalopathy

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has a hypervariable regulatory region (JCV RR). A conserved archetype form is found in the urines of healthy and immunocompromised individuals, whereas forms with tandem repeats and deletions are found in the brains of PML patients. Type I JCV RR, seen in MAD-1, the first sequenced strain of JCV, contains two 98-bp tandem repeats each containing a TATA box. Type II JCV RR has additional 23-bp and 66-bp inserts or fragments thereof and only one TATA box. We cloned and sequenced JCV RR from different anatomic compartments of PML patients and controls and correlated our findings with the patients' clinical outcome. Twenty-three different sequences were defined in 198 clones obtained from 16 patients. All 104 clones with tandem repeats were type II JCV RR. Patients with poor clinical outcome had high proportions of JCV RR clones with both tandem repeats in plasma (54%) and brain or cerebrospinal fluid (85%). In those who became survivors of PML, archetype sequences predominated in these anatomic compartments (75 and 100%, respectively). In patients with advanced human immunodeficiency virus infection without PML, only 8% of JCV RR clones obtained in the plasma contained tandem repeats. These data suggest that the presence of tandem repeats in plasma and CNS JCV RR clones is associated with poor clinical outcome in patients with PML.     

JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy.

Sections of normal and diseased brain and kidney tissues were screened for the presence of JC virus (JCV) DNA by using the polymerase chain reaction. As expected, all samples obtained from patients with progressive multifocal leukoencephalopathy (PML) tested positive when multiple JCV-specific primer and probe combinations were used. Unexpectedly, more than 50% of non-PML-affected brains were also found to harbor low levels of JCV DNA. To confirm that the positive signals seen in the tissue sections were not the result of contamination, amplified DNA was cloned and sequenced and in some cases was shown to represent strains of JCV not identified previously. Two predominant regulatory region configurations of JCV have been detected in the human host: archetype JCV, which is excreted in the urine of normal and immunocompromised individuals, and "PML-type" JCV found in diseased brains. This latter group of variants appears to derive from archetype JCV by the deletion and duplication of sequences within the promoter-enhancer region. In the present study, the archetype strain of JCV was identified only in normal kidney samples; JCV DNA found in non-PML-affected brain specimens and in kidney tissue from patients with PML resembled that of strains isolated from PML-affected brain tissue. Our findings indicate that JCV reaches the brain more frequently than previously thought and may persist at this site without causing demyelinating disease. A subsequent episode of prolonged immunodeficiency or a direct interaction with an immunocompromising agent (e.g., human immunodeficiency virus type 1) might activate the latent JCV infection and lead to the development of PML.     

Synthetic antibodies and peptides recognizing progressive multifocal leukoencephalopathy-specific point mutations in polyomavirus JC capsid viral protein 1

Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV.     

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.