Varicella zoster virus is not a disease-relevant antigen in multiple sclerosis

Abstract

Herpesvirions and varicella zoster virus (VZV) DNA were recently reported in all 15 cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (MS) obtained within 1 week of exacerbation. Using identical electron microscopic and polymerase chain reaction techniques, including additional primer sets representing different regions of the VZV genome, we found no herpesvirions or VZV DNA in MS CSF or acute MS plaques. Although enzyme-linked immunosorbent assay analysis demonstrated a higher titer of VZV antibody in MS CSF than in inflammatory control samples, recombinant antibodies prepared from clonally expanded MS CSF plasma cells did not bind to VZV. VZV is not a disease-relevant antigen in MS.

Fig 1

Analysis of human cerebrospinal fluid (CSF) and varicella zoster virus (VZV)-infected cells for VZ virions and DNA. Transmission electron microscopy was used to analyze ultracentrifuged pellets obtained from VZV-infected cells in tissue culture (1, 2) and in CSF from patients with multiple sclerosis (3, 4) and clinically isolated syndromes (CIS) (5–7). After ultracentrifugation, pellets were adsorbed onto 300-mesh grids, shadowed with 1% uranyl acetate, and visualized at 23,000× magnification. Note VZ virions in pellets of supernatants from VZV-infected cells in tissue culture (1, 2), but not after ultracentrifugation of CSFs from relapsing-remitting multiple sclerosis patients collected within 8 days of exacerbation (3, 4) or in CSF of patients during an acute CIS (5–7). Scale bar = 100nm.

Fig 2

Analysis of human cerebrospinal fluid (CSF) and recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in multiple sclerosis (MS) cerebrospinal fluid (CSF) for varicella zoster virus (VZV) reactivity. (A) Enzyme-linked immunosorbent assay (ELISA) analysis of human CSF binding to VZV. CSF samples from patients with relapsing-remitting multiple sclerosis (RRMS) and other inflammatory neurological diseases (OIND) were diluted to IgG concentrations of 10μg/ml and reacted with VZV-infected cell lysates. The eight OIND were VZV vasculopathy, cryptococcal meningitis, chronic meningitis, subacute sclerosing panencephalitis (SSPE) (2), paraneoplastic syndrome, neurosyphilis, and meningoencephalitis. Panel shows the average reactivity (± SEM) of MS CSF compared to average reactivity of OIND. (B) Immunoblotting of MS IgG with VZV. Lysates of VZV-infected (+) or uninfected (−) MeWo cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitro-cellulose membranes, and reacted with 5μg/ml IgG from MS CSF, rabbit anti-VZV, or OIND IgG. Positive control anti-VZV monoclonal antibody (M anti-VZV), polyclonal rabbit anti-VZV IgG antibody (R anti-VZV), and recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in MS CSF (5μg/ml) were used to immunoblot VZV-infected and uninfected cell lysates. All rAbs were negative (lanes I-XI). Molecular size is given in kilodaltons. (C) Immunostaining. VZV-infected (a) and un-infected (b) MeWo cells were stained with rabbit anti-VZV IgG. The same MS CSF rAbs used in immunoblotting were pooled into nine groups of three to five rAbs (5μg/ml of each rAb) and used to stain VZV-infected MeWo cells. Representative staining is indicated in panels c through f. Note overall lack of reactivity of rAbs with VZV. Bar = 100μm.