Research paperAnalysis of neutralizing antibodies to therapeutic interferon-beta in multiple sclerosis patients: A comparison of three methods in a large Australasian cohort
Introduction
Interferon-beta (IFNβ) is a first-line treatment in relapsing–remitting multiple sclerosis (MS), significantly reducing relapse rates, lesion load and disability (PRISMS Study Group, 2001, Panitch et al., 2002, Frank et al., 2004). Neutralizing antibodies to IFNβ (NAB) may develop within months of commencing therapy; they are a subset of anti-IFNβ antibodies that prevent binding of IFNβ to its receptor and the subsequent biological effects (Deisenhammer et al., 1999, Bertolotto et al., 2003, Pachner et al., 2003b, Gilli et al., 2004) resulting in a loss of clinical efficacy of IFNβ (PRISMS Study Group, 2001, Malucchi et al., 2004, Petkau et al., 2004).
NAB are measured by quantifying the inhibition of IFNβ bioactivity by patient sera in responsive cell lines such as the lung carcinoma-derived epithelial line A549 or the HeLa-derived Wistar Institute Susan Hayflick (WISH) cells. The cytopathic effect (CPE) assay for NAB recommended by the World Health Organization (WHO) measures inhibition of the antiviral activity of IFNβ by patient serum (World Health Organisation, 1985). Antiviral assays require the use of a potentially biohazardous reagent and are subject to high inter-assay variability (Nestaas et al., 1996). Due to the indirect nature of the CPE assay, controls for viral and antiviral activity of the sample must be performed in conjunction with the test for IFNβ-neutralizing activity.
The MxA assay for NAB measures serum inhibition of the type I interferon-specific induction of the MxA protein by IFNβ (Files et al., 1998, Pungor et al., 1998, Pawlowski et al., 2003). This is a nested procedure in which MxA induction in cells cultured in the presence of IFNβ and patient serum is quantified by an enzyme-linked immunosorbent assay (ELISA) of cell lysates. The MxA assay avoids the variability and safety concerns associated with antiviral assays. The assay does not require a control for endogenous viral activity, and is not confounded by antiviral factors (other than type I interferon) in the sera. However, the variability inherent in cell-based assays remains (Deisenhammer et al., 2004, Vartanian et al., 2004).
A third approach to determining the bioactivity of IFNβ is to measure the induction of IFNβ-responsive genes in the patient's cells following IFNβ injection. MxA mRNA and protein induction in peripheral blood leukocytes by IFNβ is significantly impaired or abolished in NAB-positive individuals (Deisenhammer et al., 1999, Bertolotto et al., 2003, Pachner et al., 2003a). All relapsing–remitting MS patients in a recent study showed MxA induction at the initiation of IFNβ therapy, suggesting that lack of a cellular response to IFNβ occurs as a result of continued therapy rather than as an inherent non-responsiveness to IFNβ (Gilli et al., 2005). Regular monitoring of MxA mRNA levels may prove a useful biomarker of the ability to respond to IFNβ in vivo.
In this study, we have compared the CPE and MxA in vitro assays and the MxA mRNA ex vivo assay for analysis of NAB and their effect on IFNβ bioactivity in multiple sclerosis patients in Australia and New Zealand (Australasia).
Section snippets
Samples and reagents
For NAB in vitro assays, serum samples were obtained from MS patients at least 24 h after the last IFNβ injection. The number of individuals tested by CPE and MxA assays was 227 and 350 respectively. Control sera from 20 volunteer donors were obtained from the Australian Red Cross Blood Service. Whole blood for MxA mRNA IFNβ in vivo bioactivity testing was obtained from 13 MS patients 12–24 h after the last IFNβ injection, and from 20 IFNβ-naïve control volunteers. Reference human anti-human
CPE assay results
Of 227 individuals tested for NAB by the CPE assay, 35 (15%) were NAB-positive with a range of titres from 20 to > 7290. The log10 titre of the NIH antiserum was 2.92 ± 0.11 against IFNβ-1a (n = 7 determinations) which is within the stated range for titre against human fibroblast IFNβ (3.24 ± 0.34; Research reference reagent note No. 45, NIAID, NIH, Maryland, USA).
Intra- and inter-assay variation was monitored using a NAB-positive rabbit antiserum, included in every assay. Only assays with an
Discussion
It is now widely accepted that NAB reduce the in vivo bioactivity and therapeutic effect of IFNβ. While the CPE assay is the WHO standard for NAB measurement, the MxA assay is now used by some investigators (Vartanian et al., 2004). We compared these in vitro techniques for the quantification of NAB in large cohorts of patients receiving IFNβ therapy. While results from the two assays were highly correlated and generated similar ranked titre distributions, the MxA assay showed higher sensitivity
Acknowledgements
We would like to thank Biogen-Idec for providing IFNβ, rabbit polyclonal anti-IFNβ antibodies and monoclonal anti-MxA antibodies, and Dr. Vijay Jethwa, Dr. Susan Goelz and Miki Pawlowksi (Biogen-Idec, USA) for their invaluable technical assistance. This project was supported by an education grant from Biogen-Idec.
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