Study concept and design: R. antiviral and antibacterial antibody reactivities. Peripheral JCV- and BKV-specific antibody reactions persisted, and no cross-reactivity between JCV- and BKV-specific CSF antibodies was found. Intrathecal production of antibodies against measles, rubella, and zoster antigens (MRZ reaction) was most common and persisted (73.3% before vs 66.7% after 1 year of NAT therapy). CSF OCBs also persisted (93.3% vs 80.0%), but total CSF IgG and IgM levels declined significantly. Conclusions These data show that JCV-specific antibodies are produced intrathecally inside a minority of individuals with MS, and NAT treatment affects the intrathecal humoral immune response against JCV relatively specifically compared with additional neurotropic viruses. Further studies are needed to determine whether this effect translates to higher risk of progressive multifocal leukoencephalopathy development. Natalizumab (NAT) is definitely a highly efficacious treatment for relapsing-remitting MS (RRMS) and hinders immune cells from access into the CNS. However, this also compromises physiologic CNS immune surveillance and is an important reason for NAT-associated progressive multifocal leukoencephalopathy (NAT-PML). PML is an opportunistic CNS illness with JC polyomavirus (JCV) and offers emerged as a serious adverse event in 778 NAT-treated individuals worldwide (incidence of 4.2/1,000) with fatal outcome in 23% of cases,1,2 whereas other CNS infections occur far less frequently (incidence of herpes virus CNS infections at 0.2/1,000).3 Why NAT-treated individuals are more prone to develop PML than additional viral CNS infections is not obvious.4 NAT impairs access of B and plasma cells into the CNS and is associated with decreased total CSF IgG and IgM levels.5,C7 Moreover, NAT may reduce the prevalence, quantity, and/or intensity of CSF oligoclonal bands (OCBs),7,C9 whereas the influence on polyspecific intrathecal antibody reactivity to measles, rubella, and zoster (MRZ reaction) remains less examined.7 Positive OCBs and MRZ reaction in CSF are common, well established, and stable surrogates of ongoing intrathecal humoral immune reaction in MS.10,11 Intrathecal production of JCV-specific antibodies has been JNJ-39758979 described in 3.6% of individuals with MS12 and thus may be part of the polyspecific CSF humoral immune reaction, which is usually depicted from the MRZ reaction, but which extends to many other viruses.12,C15 Of interest, JCV-specific CSF antibody production was not found in NAT-treated patients with MS, but in 55% of patients with NAT-PML,16 indicating that it is an important aspect of protective immune responses against PML. Here, we tackled how NAT influences the spectrum of intrathecally produced antibodies against numerous pathogens including JCV. Methods Individuals and samples JNJ-39758979 Combined serum and CSF samples were collected from 15 individuals (male/female percentage 1:4; mean age 40.13 years [range 18C53 years]) diagnosed with RRMS relating to 2005 revised McDonald criteria17 in the University Medical Center Hamburg-Eppendorf, Hamburg, Germany, before NAT (V0), and after 12 months (V12) and 24 months (V24, only from 6 patients, loss of follow-up in 9 patients due to denial of further participation) of NAT treatment (300 mg IV every 4 weeks). The individuals had not received steroids 4 weeks before NAT treatment or any immunomodulatory or immunosuppressive agent 3 months before NAT treatment. Additional 25 serum samples from healthy donors were utilized for determining the cutoff value (COV) of seropositivity in JCV ELISA. Standard protocol approvals, registrations, and patient consents The local ethics committee (Ethik-Kommission der ?rztekammer Hamburg, Germany, protocol No. 2758) authorized the study. All individuals gave written educated consent. Antibody detection in serum and CSF Antibody reactivity against the following viral lysates and bacterial antigens was tested using commercial ELISA packages from Euroimmun (Lbeck, Germany): measles, rubella, mumps, herpes simplex virus type 1/2 (HSV-1/HSV-2), cytomegalovirus (CMV), varicella zoster disease (VZV), influenza JNJ-39758979 disease A and B (influenza A and B), and enterovirus lysates; Epstein-Barr disease (EBV) nuclear antigen-1 (EBNA-1) and EBV viral capsid antigen (EBV-VCA); and tetanus toxoid (tetanus), diphtheria toxoid (diphtheria), Mertk antigens. Antipolyomaviral IgG reactivity was identified using BK polyomavirus (BKV)-, KI polyomavirus (KIV)-, or WU polyomavirus (WUV)-viral protein 1 (VP1) fused to glutathione S-transferase (kind gift from Robert L. Garcea, Boulder, CO) or JCV-VP1 (MAD1 strain, Life Technology Incubator, Bonn, Germany), as explained previously.18,19 Briefly, ELISA plates were coated with 200 ng of the respective polyomavirus-VP1/well and incubated with serum (1:400, 1:2,020 dilutions) and CSF (1:2, 1:10, 1:100, and 1:1,000 dilutions). Human being IgG was recognized using biotin-conjugated anti-human mouse Fc antibody and avidin horseradish peroxidase (eBioscience, Frankfurt, Germany). Optical denseness at 450 nm (OD450) was assessed using a Synergy H1 reader (BioTek, Luzern, Switzerland). Antibody reactivity was assessed in arbitrary devices (AUs) using a standard curve.