ARTICLE OPEN ACCESS

Effects of natalizumab therapy on intrathecalantiviral antibody responses in MSFabienne Largey PhD Ivan Jelcic PhD Mireia Sospedra PhD Christoph Heesen MD RolandMartin MD and

Ilijas Jelcic MD

Neurol Neuroimmunol Neuroinflamm 20196e621 doi101212NXI0000000000000621

Correspondence

Dr Ilijas Jelcic

IlijasJelcicuszch

AbstractObjectiveTo investigate the effects of natalizumab (NAT) treatment on intrathecally produced antiviralantibodies in MS

MethodsWe performed a longitudinal observational study analyzing both serum and CSF samplescollected before and during NAT treatment for antibodies against measles rubella mumpsinfluenza entero herpes and polyoma viruses including JC polyomavirus (JCV) and itsnearest homologue BK polyomavirus (BKV) and bacterial control antigens by ELISA todetermine the antigen-specific CSF antibody index (CAI) CAI ge15 indicated intrathecalsynthesis of antigen-specific antibodies Oligoclonal bands (OCBs) by isoelectric focusing andtotal IgG IgM and IgA by immunonephelometry were analyzed additionally

ResultsIntrathecal synthesis of JCV- and BKV-specific IgG was detected in 20 of patients with MS atbaseline and was lost significantly more frequently during NAT treatment compared with otherintrathecal antiviral and antibacterial antibody reactivities Peripheral JCV- and BKV-specificantibody responses persisted and no cross-reactivity between JCV- and BKV-specific CSFantibodies was found Intrathecal production of antibodies against measles rubella and zosterantigens (MRZ reaction) was most prevalent and persisted (733 before vs 667 after 1 yearof NAT therapy) CSFOCBs also persisted (933 vs 800) but total CSF IgG and IgM levelsdeclined significantly

ConclusionsThese data indicate that JCV-specific antibodies are produced intrathecally in a minority ofpatients with MS and NAT treatment affects the intrathecal humoral immune response againstJCV relatively specifically compared with other neurotropic viruses Further studies are neededto determine whether this effect translates to higher risk of progressive multifocal leukoence-phalopathy development

From the Neuroimmunology and Multiple Sclerosis Research Section (FL Ivan Jelcic MS RM Ilijas Jelcic) Department of Neurology University Hospital of Zurich Switzerlandand Institute for Neuroimmunology and Multiple Sclerosis (inims) (CH) Center for Molecular Neurobiology (ZMNH) University Medical Center Hamburg-Eppendorf HamburgGermany

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Natalizumab (NAT) is a highly efficacious treatment forrelapsing-remitting MS (RRMS) and hinders immune cellsfrom entry into the CNS However this also compromisesphysiologic CNS immune surveillance and is an importantreason for NAT-associated progressive multifocal leu-koencephalopathy (NAT-PML) PML is an opportunisticCNS infection with JC polyomavirus (JCV) and has emergedas a serious adverse event in 778 NAT-treated patientsworldwide (incidence of 421000) with fatal outcome in23 of cases12 whereas other CNS infections occur far lessfrequently (incidence of herpes virus CNS infections at 021000)3 Why NAT-treated patients are more prone to de-velop PML than other viral CNS infections is not clear4

NAT impairs entry of B and plasma cells into the CNS and isassociated with decreased total CSF IgG and IgM levels5ndash7

Moreover NAT may reduce the prevalence number andorintensity of CSF oligoclonal bands (OCBs)7ndash9 whereas theinfluence on polyspecific intrathecal antibody reactivity tomeasles rubella and zoster (MRZ reaction) remains less ex-amined7 Positive OCBs and MRZ reaction in CSF are com-mon well established and stable surrogates of ongoingintrathecal humoral immune reaction in MS1011 Intrathecalproduction of JCV-specific antibodies has been described in36 of patients with MS12 and thus may be part of the ldquopol-yspecificrdquo CSF humoral immune reaction which is usuallydepicted by the MRZ reaction but which extends to manyother viruses12ndash15 Of interest JCV-specific CSF antibodyproduction was not found in NAT-treated patients with MSbut in 55 of patients with NAT-PML16 indicating that it is animportant aspect of protective immune responses against PML

Here we addressed how NAT influences the spectrum ofintrathecally produced antibodies against various pathogensincluding JCV

MethodsPatients and samplesPaired serum and CSF samples were collected from 15patients (malefemale ratio 14 mean age 4013 years [range18ndash53 years]) diagnosed with RRMS according to 2005 re-vised McDonald criteria17 at the University Medical CenterHamburg-Eppendorf Hamburg Germany before NAT (V0)and after 12 months (V12) and 24 months (V24 only from6 patients loss of follow-up in 9 patients due to denial offurther participation) of NAT treatment (300 mg IV every 4weeks) The patients had not received steroids 4 weeks before

NAT treatment or any immunomodulatory or immunosup-pressive agent 3 months before NAT treatment Additional 25serum samples fromhealthy donors were used for determiningthe cutoff value (COV) of seropositivity in JCV ELISA

Standard protocol approvals registrationsand patient consentsThe local ethics committee (Ethik-Kommission derArztekammer Hamburg Germany protocol No 2758) ap-proved the study All patients gave written informed consent

Antibody detection in serum and CSFAntibody reactivity against the following viral lysates andbacterial antigens was tested using commercial ELISA kitsfrom Euroimmun (Lubeck Germany) measles rubellamumps herpes simplex virus type 12 (HSV-1HSV-2) cy-tomegalovirus (CMV) varicella zoster virus (VZV) influenzavirus A and B (influenza A and B) and enterovirus lysatesEpstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) andEBV viral capsid antigen (EBV-VCA) and tetanus toxoid(tetanus) diphtheria toxoid (diphtheria) Helicobacter pyloriand Chlamydophila pneumoniae antigens AntipolyomaviralIgG reactivity was determined using BK polyomavirus (BKV)-KI polyomavirus (KIV)- or WU polyomavirus (WUV)-viralprotein 1 (VP1) fused to glutathione S-transferase (kind giftfrom Robert L Garcea Boulder CO) or JCV-VP1 (MAD1strain Life Science Incubator Bonn Germany) as describedpreviously1819 Briefly ELISA plates were coated with 200 ngof the respective polyomavirus-VP1well and incubated withserum (1400 12020 dilutions) and CSF (12 110 1100and 11000 dilutions) Human IgG was detected using biotin-conjugated anti-human mouse Fc antibody and avidin horse-radish peroxidase (eBioscience Frankfurt Germany) Opticaldensity at 450 nm (OD450) was assessed using a Synergy H1reader (BioTek Luzern Switzerland) Antibody reactivity wasassessed in arbitrary units (AUs) using a standard curveobtained from a serial dilution of the respective standard serumfor interpolating ODs by 4 parameter logistic curve fit AUswithin the linear part of the standard curve were multiplied bythe corresponding dilution factor to obtain absolute AUHighly reactive samples exceeding the linear limits of thestandard curve were further diluted

The antigen-specific CSFserum antibody index (CAIspec) wascalculated according to Reiber20 Briefly CAIspec was assessedas CAIspec = QspecQIgG if QLim gt QIgG or CAI = QspecQLim

(IgG) if QLim lt QIgG The variables were calculated as followsQspec = antigen-specific IgGCSF [AU]antigen-specific IgGserum

GlossaryAU = arbitrary unit BBB = blood-brain barrier CAI = CSF antibody index CMV = cytomegalovirus COV = cutoff valueEBV = Epstein-Barr virus EBNA-1 = EBV nuclear antigen-1 EBV-VCA = EBV viral capsid antigen HSV = herpes simplexvirus JCV = JC polyomavirusMRZ reaction = measles rubella and zoster antigens NAT = natalizumabOCB = oligoclonalband OD = optical density PML = progressive multifocal leukoencephalopathy RRMS = relapsing-remitting MS VZV =varicella zoster virus

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 NeurologyorgNN

[AU] Q IgG = IgGCSFIgGserum QLim (IgG) = 093times (Qalb2

+ 6 times 10minus6)05 minus 17 times 10minus3 Qalb = albCSFalbserum (with alb =albumin) QLim (IgG) refers to the upper discrimination lineof the hyperbolic reference range for the blood-derived IgGin CSF as 0 intrathecal IgG synthesis Intrathecal antigen-specific antibody synthesis was defined as CAIspec ge 15Serum and CSF samples were preincubated with BKV beforetesting with JCV ELISA and vice versa to assess JCVBKVcross-reactivity For the determination of the COV of JCVBKV KIV and WUV seropositivity we measured sera fromthe above-mentioned patients with MS and additional 25healthy donors (among them seropositive and negativedonors) using antigen-specific ELISA Normalized ODswere calculated by subtracting the mean OD of negativecontrols and divided by the OD of 1500 diluted standardserum We determined the COV of antigen-specific sero-positivity by ranking normalized ODs and calculating theinflection point

Albumin and total IgG IgM and IgA in serum and CSF werequantified by immunonephelometry (Unilabs Zurich Swit-zerland) IgG index was calculated as IgG index = QIgGQalb

(for QIgG and Qalb see above) and IgM index and IgA indexeach accordingly IgG index ge07 indicated intrathecal syn-thesis of IgG The relative intrathecal fraction of IgM and IgArespectively (IgMIF and IgAIF) were calculated according toReiber20 IgMIF ge 10 and IgAIF ge 10 indicated significantintrathecal synthesis of IgM and IgA respectively OCBs weredetected by isoelectric focusing on polyacrylamide gels andimmunoblotting using IgG-specific antibodies (Serva Ger-many Heidelberg Germany) OCBs were considered posi-tive in case of ge2 additional bands

Statistical analysisPrism v50 (GraphPad software San Diego CA) was used forstatistical analysis The McNemar test (for dichotomousmeasures) and Wilcoxon-matched pairs test (for quantitativemeasures) were used to compare between V0 and V12 Totest whether the frequency of loss of intrathecal antibodysynthesis specific for a given antigen between V0 and V12 isthe same as the frequency of loss of intrathecal antibodysynthesis specific for all other antigens between V0 and V12we used the Fisher exact test All the 20 antigens tested andrecognized by intrathecally produced antibodies were regar-ded as a priori independent of each other The primary aimwas to compare whether loss of intrathecal JCV-specific an-tibody synthesis between V0 and V12 was significantly morefrequent than the loss of intrathecal antibody synthesis spe-cific for all antigens other than JCV Probability values of p lt005 were considered significant in the primary aim A secondexploratory aim was to compare the frequency of loss of in-trathecal antibody synthesis specific for the following re-spective antigen between V0 and V12 against that for all otherrespective antigens (1) JCV against all other antigens (2)BKV (3) KIV (4) WUV (5) measles (6) rubella (7) VZV(8) mumps (9) HSV-1 (10) HSV-2 (11) CMV (12) EBV-VCA (13) EBNA-1 (14) influenza A (15) influenza B (16)

enterovirus (17) tetanus (18) diphtheria (19)H pylori (20)C pneumoniae (21) JCV or BKV and (22) positive MRZreaction In this exploratory aim probability values of p lt000227 (00522) were considered significant according toBonferroni correction for multiple comparisons

Data availabilityAnonymized data will be shared upon request by qualifiedinvestigators

ResultsChanges of total immunoglobulin levels inserum and CSF during NAT therapyWe first analyzed total IgG IgM and IgA levels in serum(figure 1 AndashC) and CSF (figure 1 DndashF) and intrathecalproduction of total IgG IgM and IgA (figure 1GndashL) in 15patients with RRMS (malefemale ratio 14 mean age 4013years [range 18ndash53 years]) before (V0) and during NATtreatment (V12) Total IgG levels in CSF and intrathecalproduction of total IgG as determined by IgG index de-creased significantly during NAT therapy (figure 1D G andJ) In addition NAT treatment significantly reduced total IgMlevels in serum and CSF intrathecal production of IgM (IgMindex) and total IgA levels in CSF (figure 1B E F and H)However the prevalence of donors showing intrathecal IgGIgM or IgA production ie IgG index ge07 or local IgM orIgA production ge10 did not change significantly duringNAT therapy (figure 1JndashL)

Heterogeneity of intrathecal production ofpathogen-specific antibodies in untreatedpatients with MSTo assess the influence of NAT therapy on antigen-specificIgG responses we analyzed serum and CSF levels as well asintrathecal production of antigen-specific antibodiesie antigen-specific CAIantigen at before treatment (V0 n =15) after 12 months (V12 n = 15) and after 24 months ofNAT treatment (V24 n = 6 MS01ndashMS06) using an array of20 viral and bacterial antigens (figure 2) The prevalence ofdonors seropositive for the respective antigens was compa-rable with data from the literature and did not change duringNAT therapy (figure e-1 linkslwwcomNXIA146) Asexpected the spectrum of antigens recognized by intrathecallyproduced IgG varied considerably among therapy-naiveindividuals (V0 figures 2 and 3A) Before NAT therapy in-trathecal production of antibodies against ge1 antigen wasdetectable in 867 of donors against ge2 antigens in 800 ofdonors (figures 2 and 3B) No intrathecal production ofantibodies reactive to any of the 20 antigens tested was ob-served in 2 donors (133MS06 andMS15 at V0 in figure 2)whereas intrathecal production of antibodies against 10antigens was found in 2 other donors (MS01 and MS07 at V0in figure 2) The antigens most frequently recognized by in-trathecally produced antibodies were measles (733 ofdonors) rubella (600) and VZV (533) followed by

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influenza B virus (467) and C pneumoniae (400) (figure3A) MRZ reaction defined as polyclonal intrathecal antibodyreactivity to ge2 of the 3 antigens measles (M) rubella (R) andzoster (Z) was positive in 733 (figure 3B) consistent withthe proportion reported in the literature1021 Intrathecalsynthesis of antibodies against mumps virus (333) tetanustoxoid (333) influenza A virus (267) HSV-1 (200)

HSV-2 (67) CMV (67) and enterovirus (67) was lessfrequent No intrathecal synthesis of antibodies reactiveagainst EBV-VCA EBNA-1 diphtheria toxoid orHpyloriwasdetectable (figures 2 and 3A) Of interest intrathecal antibodysynthesis to the polyomaviruses KIV (267) JCV (200)BKV (200) and WUV (133) was detected in sometherapy-naive patients

Figure 1 Total IgG IgM and IgA in the CSF and serum of patients with MS before and during NAT treatment

Changes of absolute levels of total IgGIgM and IgA in serum (AndashC) and CSF(DndashF) during NAT therapy are shownChanges in intrathecal production oftotal IgG IgM and IgA are shown bydepicting IgG IgM and IgA index (ratioof CSFserum antibodies normalized toratio CSFserum albumin) (GndashI) as wellas prevalence of patients with signifi-cant intrathecal synthesis of IgG IgM orIgA (JndashL) respectively as indicated byIgG index ge07 IgMIF ge10 and IgAIF

ge10 respectively before (V0) and after12 months (V12) of NAT treatment (n =15) The p value is shown in case ofstatistically significant differences Sta-tistics Wilcoxon-matched pairs test(AndashI) and McNemar test (JndashL) NAT =natalizumab

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Changes of intrathecal production of antigen-specific antibodies during NAT therapyOnly the serum levels of rubella virusndashreactive antibodiesdecreased significantly during NAT therapy but none of theones reactive to all other antigens (figure e-2 and e-3 linkslwwcomNXIA147 and linkslwwcomNXIA148) Incontrast NAT therapy reduced CSF levels of antibodies re-active to JCV BKV KIV WUV measles virus rubella virusVZV mumps virus EBV-VCA EBNA-1 influenza A virusinfluenza B virus enterovirus and tetanus toxoid but notthose reactive to HSV-1 HSV-2 CMV diphtheria toxoid Hpylori andC pneumoniae (figure 4 and figure e-2 and e-3 linkslwwcomNXIA147 and linkslwwcomNXIA148)

During NAT therapy the prevalence of donors producing therespective antigen-specific antibodies reduced by 200 inJCV and BKV respectively by 133 in KIV and influenza Bvirus respectively by 67 in 6 of 20 antigens and stayedunchanged or undetectable in 10 of 20 antigens tested (figures2 and 3A) Three donors showed intrathecal production ofantibodies reactive to both JCV and BKV before NAT ther-apy and both reactivities were lost in all 3 donors during NATtreatment although serum JCV- and BKV-specific antibodyresponses persisted (figure 4 A and B) In addition thesedonors showed a persistent positive MRZ reaction No cross-reactivity between JCV- and BKV-specific CSF antibodies wasdetected at baseline (figure e-4 linkslwwcomNXIA149)

Figure 2 Antigen spectrum recognized by intrathecally produced antibodies in patients with MS before and during NATtreatment

Antigens are divided into polyomaviral VP1 (PyV JCV BKV KIV and WUV) viral lysates of the MRZ reaction (measles [M] rubella [R] and VZV [Z]) lysates orantigens from other neurotropic viruses (mumps HSV-1 HSV-2 CMV EBV-VCA EBNA-1 influenza A influenza B and enterovirus) toxoids (tetanus anddiphtheria toxoids) and bacterial antigens (fromnon-neurotropicHpylori and fromneurotropicC pneumoniae) Black indicates intrathecal antibody synthesis(CAI ge 15) and white no antigen-specific intrathecal antibody synthesis (CAI lt 15) Samples with AUs below 5 were considered as not detectable (N)Longitudinal samples of patients with MS were available from before treatment (V0 n = 15) after 12 months (V12 n = 15) and after 24 months of NATtreatment (V24 n = 6 only MS01ndashMS06) Detection of OCBs in the corresponding serum and CSF samples andor additional bands in CSF is depicted in thebottom lines AU = arbitrary unit CAI = CSF antibody index EBNA-1 = EBV nuclear antigen-1 EBV = Epstein-Barr virus EBV-VCA = EBV viral capsid antigen HSV= herpes simplex virus JCV = JC polyomavirus MRZ =measles rubella and zoster antigen NAT = natalizumab OCB = oligoclonal band VZV = varicella zostervirus

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Figure 3 Prevalence and loss of intrathecal production of antigen-specific antibodies and OCBs before and during NATtreatment

(A) Prevalence of patients showing intrathecal production of antibodies to the antigens indicated before (V0 n = 15 dark gray bars) and after 12 months ofNAT treatment (V12 n = 15 light gray bars) (B) Prevalence of patients with positive MRZ reaction defined as intrathecal production of antibodies against atleast 2 of the 3MRZ antigens (MRZ virus) before (V0 dark gray bars) and after 12months of NAT treatment (V12 light gray bars) (upper graph) and prevalenceof patientswith intrathecal production of antibodies against at least 2 of the 20 antigens analyzed in (A) before (V0 dark gray bars) and after 12months of NATtreatment (V12 light gray bars) (lower graph) (C) Frequency of loss of intrathecal production of antibodies (CAI ge 15) reactive against JCV (black barproportion 33) in comparison to the frequency of loss of CAI ge15 specific for all other viral and bacterial antigens tested (white bar proportion 1870) (D)Prevalence of patients with positive OCBs as determined by isoelectric focusing before (V0 dark gray bars) and after 12 months of NAT treatment (V12 lightgray bars) Statistics McNemar test (A B and D) and Fisher exact test for frequency comparisons (C) In the primary aim the frequency of loss of CAI ge15reactive against JCVwas comparedwith that specific for all other antigens and a probability value of p lt 005was considered significant (C) CAI = CSF antibodyindex MRZ = measles rubella and zoster antigen NAT = natalizumab OCB = oligoclonal band

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 NeurologyorgNN

MRZ reaction did not change significantly in all other donorsduring NAT therapy (figure 3B) Intrathecal production ofantibodies against ge2 antigens persisted in all donors (figure

3B) Intrathecal antibody production against JCV was sig-nificantly more frequently lost than the intrathecal productionof antibodies reactive against all other antigens except JCV

Figure 4 JCV- BKV- KIV- andWUV-reactive antibodies in CSFand serumbefore (V0) andafter 12monthsofNAT treatment (V12)

Serum and CSF levels of antibodies reactive to JCV (A) BKV (B) KIV (C) or WUV (D) are depicted in AUs Intrathecal production of the respective antibodies isindicated by CSFserum JCV- BKV- KIV- or WUV-reactive antibody index (CAIJCV CAIBKV CAIKIV and CAIWUV) Dashed lines indicate the value changes in the 3donors with intrathecal production of JCV- and BKV- specific antibodies at V0 and loss thereof at V12 (each n = 15) Continuous horizontal lines indicate COV ofseropositivity Dotted horizontal lines indicate the COV for intrathecal antigen-specific antibody synthesis as defined by CAIspec ge 15) AU = arbitrary unit CAI= CSF antibody index COV = cut-off value JCV = JC polyomavirus NAT = natalizumab

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 7

(primary aim p = 0021 figure 3C) The same was true for thecombination of JCV and BKV (exploratory aim p = 000032)after Bonferroni correction for multiple comparisons (n =22) but not for any of the other antigens (figure e-5 linkslwwcomNXIA150)

Changes in OCBs during NAT treatmentOCBs were positive in CSF in 933 of donors before and in800 of donors during NAT therapy (figures 2 and 3D) Lossof intrathecal OCB production occurred without loss of in-trathecal production of antigen-specific antibodies and viceversa (MS13 vs MS02 MS07 MS11 and MS12 in figure 2)OCBs turned positive in 1 patient (MS01) during NATtherapy which was not accompanied by occurrence of in-trathecal production of antigen-specific antibodies (figure 2)Altogether changes in OCB positivity did not correlatewith changes in intrathecal production of antigen-specificantibodies

DiscussionHere we report a detailed analysis of the influence of NATtherapy on the intrathecal production of antiviral and inparticular anti-JCV antibodies in patients with MS and pos-sible implications for the development of NAT-PML Of note50ndash70 of patients with MS have been reported to be JCVseropositive22 Established risk factors for NAT-PML de-velopment are increased levels of anti-JCV antibodies in se-rum duration of NAT treatment longer than 2 years andprevious long-term immunosuppressive therapies22 NATalso induces an elevation of B-cell numbers in the peripheralblood which may serve as a cell pool for JCV infection anddissemination2324 NAT-induced upregulation of B-celltranscription factors may activate early JCV gene expres-sion adding to NAT-PML risk25 Occurrence of PML duringimmunomodulatory treatments and in immunodeficienciesthat primarily affect B cells indicates the importance of anti-bodies for controlling JCV426 Recently we have discoveredviral immune escape mechanisms affecting humoral andadaptive JCV-specific immunity2627 Patients with MS atonset of NAT-PML showed reduced CSF antibody responsesagainst PML-associated JCV variants which were closed afterimmune reconstitution in the CNS for overcoming JCV im-mune escape2628 However it is not yet well understood whypatients with MS with higher serum anti-JCV antibody levelsare at a higher risk of NAT-PML22 and whether the affinity ofanti-JCV antibodies andor their ability to recognize andneutralize multiple JCV variants (including PML-associatedstrains) is deficient in these individuals426 It also remainsunclear why NAT therapy is biased to PML and not to anyother infectious CNS disease

By analyzing antibody reactivities against 20 microbial anti-gens we demonstrate high interindividual variability in thereactivity of intrathecally produced antibodies against variouspathogens in therapy-naive patients withMSWe confirm that

intrathecal production of antibodies against ge2 viral antigensis a frequent feature in therapy-naive patients with MS (foundin 800) with MRZ reaction being most prevalent(733)1013ndash1521 As an interesting novel observation wedetected intrathecal production of antibodies in therapy-naivepatients with MS against tetanus toxoid (333) JCV(200) BKV (200) KIV (267) and WUV (133)This response may be part of theMS-associated ldquopolyspecificrdquoCSF antibody response similar to a MRZ-like reaction1021

What the role of this broad intrathecal humoral immune re-sponse in the pathogenesis of MS is and whether it is pro-tective against viral CNS infections are not clear

In patients with MS antibody-producing cells (memoryB cells plasmablasts and plasma cells) frequently circulatebetween CNS and peripheral blood2930 indicating a role ofperipheral humoral immunity in shaping the CNS-residentB-cell repertoire The mechanisms establishing the intrathecalB-cell repertoire are as yet unknown However MS-associatedCNS inflammation might attract antibody-producing cells bychemokine cues31 The probability that B cells producingantibodies against a specific pathogen settle to the CNSmightbe influenced by the frequency of respective CNS homingantibody-producing cells in peripheral blood and their sto-chastic migration into the CNS Additional factors may beexpression of molecules that are needed for CNS entry andbystander signals for survival

Of interest simultaneous intrathecal JCV- and BKV-specificantibody production was found in 20 of patients beforeNAT therapy but disappeared during NAT therapy in all ofthese patients with MS whereas anti-JCV and -BKV serumantibodies andMRZ reaction and CSFOCBs persisted Initialreports suggested disappearance of OCBs during NAT ther-apy in 55ndash67 of patients9 but our data support the per-sistence of OCBs during NAT therapy and corroborates morerecent reports about less prominent changes in OCB preva-lence found in 0ndash16 of NAT-treated patients78 Meth-odological differences in OCB detection between the CSFlaboratories and differences inMS populations analyzed in therespective studies could account for the discrepant OCBfindings seen Importantly JCV- and BKV-specific antibodyresponses were significantly more frequently lost than in-trathecal antibody reactivities against other neurotropicviruses and infectious agents suggesting an antigen-selectiveloss BKV shares the highest sequence identity with JCVamong human polyomaviruses and BKV-specific antibodiesfrom healthy donors show frequent cross-reactivity againstJCV26 However BKV causes nephropathy in up to 10 ofkidney transplant recipients and is not considered to be as-sociated with PML MS or brain disease

Differential CNS-migratory properties or requirement ofsurvival factors for JCV- or BKV-specific antibody-producingcells might account for this selective loss Very Late Antigen(VLA-4) expression allows antibody-producing cells to crossthe blood-brain barrier (BBB)3233 Not only VLA-4

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 NeurologyorgNN

expression levels but also the degree of ligand affinity of VLA-4 which may be modulated by the activation state of the celldetermine adhesion and migratory abilities3435 Lower ex-pression or ligand affinity of VLA-4 could be an importantfactor to hinder JCV- and BKV-specific antibody-producingcells more efficiently from crossing the BBB during NATtherapy than other pathogen-specific B cells

On CNS entry plasmablasts that occupy survival niches suc-cessfully differentiate into long-lived plasma cells and becomeimmobile31 In contrast antibody-producing cells lacking survivalcues might eventually undergo apoptosis and intrathecal anti-body secretion might depend on continuous CNS infiltration ofperipheral cells We consider this scenario rather unlikely in thecontext of JCV- and BKV-specific B cells because the heredetected absence of cross-reactivity between JCV- and BKV-specific antibodies in the CNS which is usually found in sera ofhealthy donors and patients with MS26 may indicate affinitymaturation of CNS homing initially cross-reactive B cells to JCV-monospecific and BKV-monospecific cells Alternatively in-trathecal JCV- and BKV-specific antibody-producing cells mightbe more dependent on CD4+ T-cell help for survival and mat-uration to long-lived plasma cells than B cells specific for otherpathogens Again NATmay inhibit CNS entry of antiviral CD4+

T cells differentially depending on peripheral precursor fre-quencies expression levels of VLA-4 and migratory abilities634

Whereas 20 of therapy-naive patients with MS show in-trathecal JCV- and BKV-specific antibody production NATtherapy appears to abrogate these immune reactions preferen-tially compared with responses against other neurotropic virusesThe observations of this study not only show the previouslyknown interindividual heterogeneity of the intrathecal poly-specific antiviral humoral immune reactions in patients with MSbut also indicate intraindividual heterogeneity in the persistenceof intrathecal antigen-specific antibody reactivities with somebeing more stable and persistent (eg MRZ reaction) thanothers (intrathecal JCV-specific antibody synthesis) Assumingthat intrathecally produced anti-JCV antibodies are protectivetheir loss during NAT treatment could result in higher risk forPML development Limitations of our study are the small cohortsize and short observation period respectively Larger studies areneeded to define how NAT-induced loss of intrathecal JCV-specific antibody production influences PML risk

Author contributionsF Largey and Il Jelcic had full access to all the data in thestudy and take responsibility for the integrity of the data andthe accuracy of the data analysis Study concept and design RMartin and Il Jelcic Acquisition analysis or interpretation ofdata F Largey Iv Jelcic M Sospedra RMartin and Il JelcicDrafting of the manuscript F Largey R Martin and Il JelcicCritical revision of the manuscript for important intellectualcontent Iv Jelcic M Sospedra and C Heesen Statisticalanalysis F Largey and Il Jelcic Administrative technical ormaterial support M Sospedra C Heesen R Martin and IlJelcic Study supervision Il Jelcic

AcknowledgmentThe authors thank all patients who participated in the studythe Section of Neuroimmunology and Multiple SclerosisResearch is supported by the Clinical Research PriorityProgram (CRPP-MS) of the University of Zurich TheInstitute for Neuroimmunology and Clinical MultipleSclerosis Research was supported by the GemeinnutzigeHertie Stiftung The authors thank Dr Robert L GarceaDepartment of Molecular Cellular amp Developmental Bi-ology and The Biofrontiers Institute University of Coloradoat Boulder CO for providing BKV- KIV- and WUV VP1antigens for this study They further thank Dr PatrickStellmann Department of Neurology University MedicalCenter Hamburg-Eppendorf Hamburg Germany for check-ing clinical data of the patients and Katja SteinhagenEuroimmun Lubeck Germany for technical advice They arealso indebted to Matthias Kirchner ETH Zurich ZurichSwitzerland for guidance in the statistical analysis of the data

Study fundingThis study was supported by an unrestricted grant fromBiogen Idec Inc The Section of Neuroimmunology andMultiple Sclerosis Research is supported by the Clinical Re-search Priority Program (CRPP-MS) of the University ofZurich The Institute for Neuroimmunology and ClinicalMultiple Sclerosis Research was supported by the Gemein-nutzige Hertie Stiftung Role of the fundersponsor thefunding source had no role in the design and conduct of thestudy collection management analysis and interpretation ofthe data preparation review or approval of the manuscriptand decision to submit the manuscript for publication

DisclosureF Largey reports no disclosures Iv Jelcic received compen-sation for serving on a scientific advisory board by SanofiGenzyme M Sospedra has received an unrestricted grantfrom Biogen Idec C Heesen has received speaker honorariesand research grants from Biogen Genzyme Merck Novartisand Roche R Martin has served as advisor or member ofspeakerrsquos boards for Biogen Idec Novartis Roche GenzymeCellProtect and Neuway Furthermore the group of RMartin has received unrestricted grants from Biogen Idec andNovartis Il Jelcic has received speaker honoraria or un-restricted grants from Biogen Idec and Novartis and hasserved as advisor for Neuway Merck Novartis and SanofiGenzyme Go to NeurologyorgNN for full disclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationAugust 15 2018 Accepted in final form July 31 2019

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modulatory therapies Annu Rev Med 20106135ndash472 Biogen Tysabri Safety Update Available at biogenchde_CHmedical-pro-

fessionalsrxsecuretysabrihtml Accessed on November 11 20183 Fine AJ Sorbello A Kortepeter C Scarazzini L Central nervous system herpes

simplex and varicella zoster virus infections in natalizumab-treated patients ClinInfect Dis 201357849ndash852

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 9

4 Jelcic I Jelcic I Faigle W Sospedra M Martin R Immunology of progressive mul-tifocal leukoencephalopathy J Neurovirol 201521614ndash622

5 Selter RC Biberacher V Grummel V et al Natalizumab treatment decreases serumIgM and IgG levels in multiple sclerosis patients Mult Scler 2013191454ndash1461

6 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

7 Warnke C StettnerM Lehmensiek V et al Natalizumab exerts a suppressive effect onsurrogates of B cell function in blood and CSF Mult Scler 2015211036ndash1044

8 Harrer A Tumani H Niendorf S et al Cerebrospinal fluid parameters of B cell-relatedactivity in patients with active disease during natalizumab therapy Mult Scler 2013191209ndash1212

9 Mancuso R Franciotta D Rovaris M et al Effects of natalizumab on oligoclonalbands in the cerebrospinal fluid of multiple sclerosis patients a longitudinal studyMult Scler 2014201900ndash1903

10 Stangel M Fredrikson S Meinl E Petzold A Stuve O Tumani H The utility ofcerebrospinal fluid analysis in patients with multiple sclerosis Nat Rev Neurol 20139267ndash276

11 Bonnan M Intrathecal IgG synthesis a resistant and valuable target for future mul-tiple sclerosis treatments Mult Scler Int 20152015296184

12 Weber T Trebst C Frye S et al Analysis of the systemic and intrathecal humoralimmune response in progressive multifocal leukoencephalopathy J Infect Dis 1997176250ndash254

13 Salmi A Reunanen M Ilonen J Panelius M Intrathecal antibody synthesis to virusantigens in multiple sclerosis Clin Exp Immunol 198352241ndash249

14 Felgenhauer K Schadlich HJ Nekic M Ackermann R Cerebrospinal fluid virusantibodies A diagnostic indicator for multiple sclerosis J Neurol Sci 198571291ndash299

15 Derfuss T Gurkov R Then Bergh F et al Intrathecal antibody production againstchlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune responseBrain 20011241325ndash1335

16 Warnke C von Geldern G Markwerth P et al Cerebrospinal fluid JC virus antibodyindex for diagnosis of natalizumab-associated progressive multifocal leukoencephal-opathy Ann Neurol 201476792ndash801

17 Polman CH Reingold SC Edan G et al Diagnostic criteria for multiple sclerosis2005 revisions to the ldquoMcDonald criteriardquo Ann Neurol 200558840ndash846

18 Kean JM Rao S Wang M Garcea RL Seroepidemiology of human polyomavirusesPLoS Pathog 20095e1000363

19 Aly L Yousef S Schippling S et al Central role of JC virus-specific CD4+-lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitutioninflammatory syndrome Brain 20111342687ndash2702

20 Reiber H Cerebrospinal fluidndashphysiology analysis and interpretation of proteinpatterns for diagnosis of neurological diseases Mult Scler 1998499ndash107

21 Reiber H Ungefehr S Jacobi C The intrathecal polyspecific and oligoclonal immuneresponse in multiple sclerosis Mult Scler 19984111ndash117

22 Plavina T Subramanyam M Bloomgren G et al Anti-JC virus antibody levels inserum or plasma further define risk of natalizumab-associated progressive multifocalleukoencephalopathy Ann Neurol 201476802ndash812

23 Frohman EM Monaco MC Remington G et al JC virus in CD34+ and CD19+ cellsin patients with multiple sclerosis treated with natalizumab JAMA Neurol 201471596ndash602

24 Planas R Jelcic I Schippling S Martin R Sospedra M Natalizumab treatment per-turbs memory- andmarginal zone-like B-cell homing in secondary lymphoid organs inmultiple sclerosis Eur J Immunol 201242790ndash798

25 Meira M Sievers C Hoffmann F et al Natalizumab-induced POU2AF1Spi-Bupregulation a possible route for PML development Neurol Neuroimmunol Neu-roinflamm 20163e223 doi101212NXI0000000000000223

26 Jelcic I Combaluzier B Jelcic I et al Broadly neutralizing human monoclonal JCpolyomavirus VP1-specific antibodies as candidate therapeutics for progressive mul-tifocal leukoencephalopathy Sci Transl Med 20157306ra150

27 Jelcic I Jelcic I Kempf C et al Mechanisms of immune escape in central nervoussystem infection with neurotropic JC virus variant Ann Neurol 201679404ndash418

28 Ray U Cinque P Gerevini S et al JC polyomavirus mutants escape antibody-mediated neutralization Sci Transl Med 20157306ra151

29 Palanichamy A Apeltsin L Kuo TC et al Immunoglobulin class-switched B cellsform an active immune axis between CNS and periphery in multiple sclerosis SciTransl Med 20146248ra106

30 Stern JN Yaari G Vander Heiden JA et al B cells populating the multiple sclerosisbrain mature in the draining cervical lymph nodes Sci Transl Med 20146248ra107

31 Radbruch A Muehlinghaus G Luger EO et al Competence and competition thechallenge of becoming a long-lived plasma cell Nat Rev Immunol 20066741ndash750

32 Lehmann-Horn K Sagan SA Bernard CC Sobel RA Zamvil SS B-cell very lateantigen-4 deficiency reduces leukocyte recruitment and susceptibility to central ner-vous system autoimmunity Ann Neurol 201577902ndash908

33 Alter A Duddy M Hebert S et al Determinants of human B cell migration acrossbrain endothelial cells J Immunol 20031704497ndash4505

34 Niino M Bodner C Simard ML et al Natalizumab effects on immune cell responsesin multiple sclerosis Ann Neurol 200659748ndash754

35 Hyduk SJ Oh J Xiao H Chen M Cybulsky MI Paxillin selectively associates withconstitutive and chemoattractant-induced high-affinity alpha4beta1 integrins impli-cations for integrin signaling Blood 20041042818ndash2824

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 6 | November 2019 NeurologyorgNN

DOI 101212NXI000000000000062120196 Neurol Neuroimmunol Neuroinflamm

Fabienne Largey Ivan Jelcic Mireia Sospedra et al Effects of natalizumab therapy on intrathecal antiviral antibody responses in MS

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