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Assessment of NAb Levels in MS Patients

New Assay Evaluates Neutralizing Anti-IFN-β in Individuals Afflicted with Multiple Sclerosis

• Ronald Jubin, Ph.D.

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Choosing an Assay Format

Figure 1. In this CPE assay, encephalomyocarditis (EMC) virus was used to kill the human lung carcinoma cell line A549. Cell density and EMC virus dilution were optimized to yield a good differential between the cell control (no virus) and the virus control (no IFN-β). Under these conditions, IFN-β protects the cells in a dose-dependent manner. The EC50 for IFN-β generally ranges from 0.5 to 2 units/mL. Click Image To Enlarge +

Figure 1. In this CPE assay, encephalomyocarditis (EMC) virus was used to kill the human lung carcinoma cell line A549. Cell density and EMC virus dilution were optimized to yield a good differential between the cell control (no virus) and the virus control (no IFN-β). Under these conditions, IFN-β protects the cells in a dose-dependent manner. The EC50 for IFN-β generally ranges from 0.5 to 2 units/mL.

Neutralizing antibodies to IFN-β would be expected to block the function of the cytokine. IFN-β is a secreted cellular protein that inhibits viral replication. Consequently, building a robust NAb assay actually begins with the development of a sensitive functional activity assay.

Viral challenge assays have become the primary method used to determine the amount of IFN-β in an unknown sample. Briefly, cells are exposed to the putative IFN-containing sample, incubated, and subsequently challenged with a cytopathic virus. Control cells that are challenged with a virus in the absence of IFN-β pretreatment undergo death, a process referred to as the cytopathic effect (CPE).

IFN-β pretreatment of cells prevents viral replication, thereby blocking CPE in a dose-dependent manner (Figure 1). After a sensitive antiviral assay has been developed, then anti-IFN-β antibodies produced in animals can be screened to identify candidates to be used as positive controls in NAb screening assays.

NAb Control Antibody Candidates

Figure 2. IFN-β-1a dose-dependency on the neutralizing effects of anti-IFN-β Abs (Part I): In one format, PAbs were serially diluted and screened against a constant concentration of IFN-β-1a (10 IU/mL). Several candidate PAbs were identified by this approach. All PAbs displayed neutralizing activities in a dose-dependent manner, with PAb 31410-1 displaying best-fit neutralization. Since these antibodies were produced in rabbits using highly purified IFN-β, they were not capable of inhibiting other interferons when screened in specificity assays. Likewise, the antibodies were tested alone for any effects on the antiviral assay, and they all showed no interference. Thus these antibodies, particularly 31410-1, displayed ideal characteristics for use as a NAb assay control antibody. Click Image To Enlarge +

Figure 2. IFN-β-1a dose-dependency on the neutralizing effects of anti-IFN-β Abs (Part I): In one format, PAbs were serially diluted and screened against a constant concentration of IFN-β-1a (10 IU/mL). Several candidate PAbs were identified by this approach. All PAbs displayed neutralizing activities in a dose-dependent manner, with PAb 31410-1 displaying best-fit neutralization. Since these antibodies were produced in rabbits using highly purified IFN-β, they were not capable of inhibiting other interferons when screened in specificity assays. Likewise, the antibodies were tested alone for any effects on the antiviral assay, and they all showed no interference. Thus these antibodies, particularly 31410-1, displayed ideal characteristics for use as a NAb assay control antibody.

During assay development, sensitivity, precision, and robustness are important parameters. However, the most challenging aspect of NAb assay development is preparation of a potent NAb to serve as a positive control. Since NAb activity cannot be determined a priori, the only way to evaluate candidates is through rigorous screening.

In Figure 2, a panel of polyclonal antibodies (PAb) was examined for their neutralization potential. When NAbs are present in a particular sample, the protective effects of IFN-β are blocked and CPE is observed.

Assay Precision

NAb activity and assay precision Click Image To Enlarge +

NAb activity and assay precision

Many different parameters can affect the precision and reproducibility of a biological assay. Consequently, it is important to establish a robust assay that performs consistently in the hands of multiple investigators. The Table shows the results of one such study.

The results reported in this study highlight the testing of a panel of anti-IFN-β antibodies displaying varying neutralizing capacities. Of these, PAb 31410-1 exhibited the most potent neutralization capability and lowest CV rate. Our results also demonstrated that the NAb assay is highly reproducible, and produces comparable neutralization titers even when used by different investigators.

Inter-operator reproducibility is critical, as this would allow for direct comparison of patient results between multiple operators within one or more diagnostic laboratories. These initial studies are promising and warrant further expanded studies with MS patient serum samples to better determine the assay sensitivity, precision, and false-positive rate.

Clearly, the immune response in MS patients is not uniform. Individual variations in the types of T helper cells present, and the levels and potency of neutralizing anti-IFN-β antibodies present in patients can vary greatly and potentially impact the efficacy of IFN-β therapy.

The ability to accurately predict the likelihood of a given patient response to IFN-β therapy (i.e., not in the high IL-17/IFN-β MS subtype) would be of enormous clinical benefit given the high costs and potential side effects of continuous IFN-β therapy. Thus, having an assay that precisely and reproducibly evaluates the levels of neutralizing anti-IFN-β present in patient serum samples is a critical step toward achieving this goal.