Research ArticleInfectious Disease

Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

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Science Translational Medicine  23 Sep 2015:
Vol. 7, Issue 306, pp. 306ra150
DOI: 10.1126/scitranslmed.aac8691
  • Fig. 1. VP1 mutations compromise serum antibody responses in NAT-PML.

    (A) SDS–polyacrylamide gel electrophoresis (PAGE) of monomeric recombinant VP1 variants that were used as pentamers in ELISA. (B) Serial dilutions of a human standard serum with a high concentration of JCPyV VP1–specific antibodies and equal recognition of all VP1 variants used as a reference standard to estimate antibody binding expressed as arbitrary units (AU). (C) Serum antibody responses of HDs and NAT-treated MS, NAT-PML, and NAT-PML-IRIS patients to VP1MAD1 pentamers, depicted in AUs (mean ± SEM). Statistically significant P values are shown for HD versus NAT-PML-IRIS (n = 38; P = 0.0027, Kruskal-Wallis), HD versus NAT-PML (n = 45; P = 0.0206, Kruskal-Wallis), NAT versus NAT-PML-IRIS (n = 36; P = 0.0037, Kruskal-Wallis), and NAT versus NAT-PML (n = 43; P = 0.0283, Kruskal-Wallis). (D) Relative binding of individual sera of the different donor populations to VP1 variants in comparison to VP1MAD1 (mean ± SEM). Data from each donor were normalized to its corresponding recognition to VP1MAD1 set as 100% binding (red dotted line). Statistically significant P values are shown for HD versus NAT-PML (n = 45; P = 0.0002, Kruskal-Wallis), and NAT versus NAT-PML-IRIS (n = 35; P = 0.0055, Kruskal-Wallis), and NAT-PML versus NAT-PML-IRIS (n = 23; P < 0.0001, Kruskal-Wallis) for binding to VP1L55F, and NAT versus NAT-PML-IRIS (n = 35; P = 0.0034, Kruskal-Wallis) for binding to VP1S269F. (E) Relative binding of serum from patients with NAT PML to VP1 variants in comparison to VP1MAD1, represented as a heat map. Binding efficiency to VP1 variants is illustrated by color gradient. Data were normalized as in (D).

  • Fig. 2. Immune reconstitution in NAT-PML enhances intrathecal antibody response against VP1 variants.

    (A) CSF antibody responses of NAT-PML and NAT-PML-IRIS patients to VP1MAD1 pentamers, depicted in AUs (mean ± SEM). Statistically significant P value is shown (n = 22; P = 0.0022, Mann-Whitney U test). (B) Relative binding of CSF from each individual at diagnosis of PML or onset of PML-IRIS to VP1 variants in comparison to VP1MAD1 (mean ± SEM). Data from each patient were normalized to its corresponding recognition to VP1MAD1 set as 100% binding (red dotted line). Statistically significant P value is shown for binding to VP1L55F (n = 22; P = 0.0106, Mann-Whitney U test). (C) Intrathecal antibody production against VP1 variants in PML and PML-IRIS was determined by calculation of CAI VP1JCPyV, which accounts for VP1-specific antibody levels in the serum and CSF normalized to total albumin and total IgG CSF/serum ratio. A CAI VP1JCPyV >1.5 (red dotted line) was considered as evidence for intrathecal antibody synthesis. Statistically significant P values are shown for CAI VP1MAD1 (n = 22; P = 0.0081, Mann-Whitney U test), for CAI VP1WT3 (n = 22; P = 0.0061, Mann-Whitney U test), for CAI VP1L55F (n = 22; P = 0.0103, Mann-Whitney U test), for CAI VP1S267F (n = 22; P = 0.0159, Mann-Whitney U test), and for CAI VP1S269F (n = 22; P = 0.024, Mann-Whitney U test).

  • Fig. 3. Human-derived monoclonal antibodies against JCPyV VP1 show distinct binding characteristics with respect to their affinity and cross-reactivity against BKV VP1.

    Human-derived JCPyV VP1–specific monoclonal antibodies were recombinantly cloned from HDs and a NAT-PML-IRIS patient and characterized with respect to their binding properties.(A to C) Examples of high-affinity JC VLP–specific antibody (98D3) targeting a conformational epitope, (B) JC/BK VLP cross-reactive antibody (44F6B) targeting a conformational epitope, and (C) JC/BK VLP cross-reactive antibody (11G6) targeting a linear epitope. Affinity and BKPyV cross-reactivity were determined by EC50 in a serial dilution of JC/BK VLP ELISA. ELISA of native and denatured JCPyV VLP and Western blot of JCPyV VP1 and BKPyV VP1 were used to characterize recognition of linear or conformational epitopes. mAbs, monoclonal antibodies.

  • Fig. 4. Identification of neutralizing JCPyV-specific human monoclonal antibodies.

    (A) Neutralization assay using JCPyVMAD4 on SVG-A cells. An appropriate dilution of the JCPyVMAD4 strain was incubated for 1 hour in the absence or presence of human monoclonal antibodies or isotype control at 50 μg/ml. Infected cells were detected by staining with a mouse anti–JCPyV VP1 (green) 72 hours after infection. The nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. Representative images illustrating the neutralization capacity of antibodies belonging to the different categories are shown. (B) Binding affinities represented as EC50 value of HD-derived (left graph) and NAT-PML-IRIS patient–derived (right graph) JCPyV VP1–specific monoclonal antibodies against JCPyV and BKPyV VLP. Antibodies were classified as non–neutralizing (blue) or neutralizing (green).

  • Fig. 5. Human monoclonal antibodies display diverse binding profiles to JCV VP1 variants.

    (A and B)Relative binding of NAT-PML-IRIS patient–derived monoclonal antibodies to VP1 variants in comparison to VP1MAD1 depicted as a heat map (A) or scatter plot (mean ± SEM) (B). Data for each antibody were normalized to its corresponding recognition to VP1MAD1 set as 100% binding (red dotted line in B). The antibodies were arranged according to their affinities for JCPyV VLP, and JCPyV neutralization capacity as well as BKPyV cross-reactivity are indicated. (B) Gray-filled circles represent individual monoclonal antibodies and red filled circles reflect serum antibody responses of the NAT-PML-IRIS patient, from whom the antibodies were cloned. (C) Relative binding of different HD-derived monoclonal antibodies to VP1 variants in comparison to VP1MAD1, represented as a heat map. Binding efficiency to VP1 variants is illustrated with a color gradient. nd, not determined.

  • Fig. 6. Phylogeny of IGH sequences of NAT-PML-IRIS patient–derived monoclonal antibodies as inferred by maximum parsimony.

    The branch length represents the amount of evolutionary divergence.

  • Table 1. Demographic data of patients and controls.

    Age and duration of NAT treatment are shown for NAT-treated MS, NAT-PML, and NAT-PML-IRIS patients and HDs. No statistically significant differences in age, female to male ratio, and duration of NAT treatment were detected between the groups.

    NATNAT-PMLNAT-PML-IRISHD
    Number (n)2815830
    Mean age (years ± SD)37.6 ± 9.238.1 ± 8.342.0 ± 6.633.9 ± 7.5
    Range of age (years)19–5323–5532–5525–49
    Female to male ratio1.82.01.71.5
    Mean duration of NAT treatment (months ± SD)42.9 ± 22.051.1 ± 16.349.8 ± 11.3
    Range of treatment duration (months)3–8614–8135–72
  • Table 2. IGH gene usage of NAT-PML-IRIS patient–derived monoclonal antibodies.

    The germline usage for heavy-chain variable region (VH), heavy-chain diversity region (DH), and heavy-chain joining region (JH) segments of IGH genes defined using the IMGT database and the number of amino acids in the CDR3 region as well as amino acid mutations as compared to the germline sequence are shown. aa, amino acids.

    mAbVHDHJHCDR3 lengthaa mutations
    98D33-30*145-5*014-1*021613/96
    43E83-30*043-22*014-1*021419/95
    105C73-23*042-15*014-1*021914/96
    45E103-23*042-15*014-1*021913/96
    29B13-23*043-22*014-1*021717/96
    26A33-23*043-3*026-1*021713/96
    58C73-11*016-13*014-1*021610/96
    47B113-11*016-13*014-1*02165/96
    56A83-21*011-1*014-1*02113/96
    27C23-21*011-1*014-1*02118/96
    72F73-48*033-3*016-1*02215/96
    59A73-48*035-5*014-1*01164/96
    57D43-9*014-17*016-1*02147/96
    105A63-9*015-5*016-1*02142/96
    72F103-9*015-12*014-1*021410/96
    27C114-4*021-14*016-1*02205/96
    50H44-4*021-14*016-1*02206/96
    7J34-30.4*013-10*016-1*02203/97
    98H14-31*033-22*016-1*022111/97
    53B115-51*011-26*014-1*02133/96

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/306/306ra150/DC1

    Materials and Methods

    Fig. S1. Mutations in JCPyV viral capsid protein VP1.

    Fig. S2. Recognition of JCPyV VP1/VLP variants by human monoclonal antibodies.

    Fig. S3. Cross-competition assay of VP1-specific monoclonal antibodies.

    Fig. S4. Differences between JCPyV and BKPyV VP1.

    Fig. S5. Recognition of denatured JCPyV VP1 and BKPyV VP1 (Western blot).

    Source data in tabular form (separate Excel file)

  • Supplementary Material for:

    Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    Ivan Jelcic, Benoit Combaluzier, Ilijas Jelcic, Wolfgang Faigle, Luzia Senn, Brenda J. Reinhart, Luisa Ströh, Roger M. Nitsch, Thilo Stehle, Mireia Sospedra, Jan Grimm,* Roland Martin*

    *Corresponding author. E-mail: roland.martin{at}usz.ch (R.M.); jan.grimm{at}neurimmune.com (J.G.)

    Published 23 September 2015, Sci. Transl. Med. 7, 306ra150 (2015)
    DOI: 10.1126/scitranslmed.aac8691

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Mutations in JCPyV viral capsid protein VP1.
    • Fig. S2. Recognition of JCPyV VP1/VLP variants by human monoclonal antibodies.
    • Fig. S3. Cross-competition assay of VP1-specific monoclonal antibodies.
    • Fig. S4. Differences between JCPyV and BKPyV VP1.
    • Fig. S5. Recognition of denatured JCPyV VP1 and BKPyV VP1 (Western blot).

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Source data in tabular form (separate Excel file)

    [Download Source Data]

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