Neutralizing antibodies to interferon beta-1a and interferon beta-1b in MS patients are cross-reactive.

OBJECTIVE: To determine whether neutralizing antibodies (NABs) to interferon beta (IFNbeta)-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react. BACKGROUND: A total of 38% of MS patients treated with IFNbeta-1b and 22% of those treated with IFNbeta-1a were reported to develop NABs, which could reduce the clinical efficacy of the drug. METHODS: Blood from 10 MS patients was collected before and at 3 and 6 months after initiating treatment with IFNbeta-1a. ELISA was performed to detect binding antibodies to IFNbeta-1a. Sera from patients who tested positive for binding antibodies to IFNbeta-1a were then screened for NABs to IFNbeta-1a in a biologic assay based on neutralization of antiviral activity. These serum samples were subsequently tested for cross-reactivity with IFNbeta-1b both in the ELISA and the biologic assay. In the second part of the study, sera from patients who participated in the phase III IFNbeta-1b trial at the University of Maryland were examined for cross-reactivity with IFNbeta-1a in the ELISA and the biologic assay. RESULTS: Of the 10 patients treated with IFNbeta-1a, three developed binding as well as NABs to IFNbeta-1a 6 months after treatment, and these antibodies cross-reacted with IFNbeta-1b both in the binding and the biologic assay. Similarly, sera from six patients with NABs to IFNbeta-1b showed cross-reactivity with IFNbeta-1a in the binding assay. Three of these six serum samples tested for neutralizing activity against IFNbeta-1a demonstrated the presence of NABs to IFNbeta-1a. CONCLUSIONS: NABs to IFNbeta-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react, both in the binding and the biologic assays. This suggests that switching to alternate IFNbeta preparation in patients who develop NABs may not be clinically beneficial. Studies examining cross-reactivity between NABs to IFNbeta-1a and IFNbeta-1b in a large number of patients are indicated.

Serum interferon beta-1a (Avonex) levels following intramuscular injection in relapsing-remitting MS patients.

BACKGROUND: The pharmacokinetics of IFNbeta-1a in MS patients are poorly understood. We have previously reported an ELISA sensitive and specific for measuring serum IFNbeta-1b levels in patients with MS. OBJECTIVE: We describe an ELISA to measure interferon beta-1a (Avonex) in the serum of MS patients following IM administration. METHODS: We have developed an ELISA for detecting serum IFNbeta-1a in MS patients receiving 6 million units (MU) of IFNbeta-1a, IM once weekly. The specificity of this ELISA was confirmed by the lack of cross-reactivity with other cytokines except for IFNbeta-1b. RESULTS: Serum IFNbeta-1a levels were measured at 3 and 6 months after initiating treatment with IFNbeta-1a in 10 MS patients. At 3 months, all 10 patients had detectable levels ranging from 68 to 86 IU/mL. At 6 months, IFNbeta-1a could be detected in the serum of all but three patients, with levels ranging from 64 to 81 IU/mL. A kinetic study of IFNbeta-1a serum levels in a separate group of six MS patients who had been receiving IFNbeta-1a for several months was carried out. Blood was drawn before and 2, 4, 6, 8, and 24 hours after IFNbeta-1a injection. Peak serum IFNbeta-1a levels were observed at 8 hours and became undetectable at 24 hours after injection. CONCLUSION: The described ELISA may have useful clinical applications in examining the correlation between serum IFNbeta-1a levels and clinical efficacy.

Immunomodulating functions of recombinant ovine interferon tau: potential for therapy in multiple sclerosis and autoimmune disorders.

The interferons (IFN) are a family of complex proteins possessing antiviral, antiproliferative, and immunomodulatory activities. Two type I recombinant human IFN have been recently approved for the treatment of multiple sclerosis (MS). However, use of high dose type I IFN treatment in MS patients has been limited by dose-related toxicity. Ovine IFN tau is a unique type I interferon discovered for its role in the animal reproductive cycle. It differs from other type I IFNs in that it is remarkably less toxic even at high concentrations, is able to cross species barriers, and is not inducible by viral infection. Ovine IFN tau has been shown to be very effective in the treatment of animal models of MS. In this study, we examined the toxicity of OvIFN tau on human T-cells at high doses and its immunregulatory properties at equivalent doses. Our experiments confirmed the remarkably non-toxic nature of OvIFN tau on human cells at high concentrations as well as immunomodulating properties consistent with other type I IFNs including an antilymphoproliferative effect and inhibition of IFN gamma-induced HLA class II expression. These results suggest that OvIFN tau could be developed into a potentially less toxic therapeutic option for immune-mediated disorders including MS.

Interferon beta-1b serum levels in multiple sclerosis patients following subcutaneous administration.

Recombinant interferon beta-1b (rIFNbeta) reduces the frequency of exacerbations in relapsing-remitting MS when administered subcutaneously on alternate days. However, the pharmacokinetics of rIFNbeta are not well understood and there are scant data on the detectability of rIFNbeta in the serum of MS patients following subcutaneous administration. Moreover, existing assays for detecting IFNbeta are biologic, time-consuming, and require handling of infectious agents. We developed and standardized an ELISA specific for measuring rIFNbeta with a detection range of 40 to 1,000 IU/ml. The specificity of our ELISA was confirmed by the lack of cross-reactivity with other cytokines, including IFNalpha, IFNgamma, IFN Consensus-1, and TNFalpha. We screened serum from 34 MS patients drawn within 12-36 hours of treatment: 15 patients taking 8 MIU, four patients taking 1.6 MIU, and 15 patients taking placebo. Eleven of the 15 patients in the 8-MIU treatment group had measurable rIFNbeta serum levels ranging from 120 to 475 MIU/ml. Two of four patients in the 1.6-MIU treatment group, but none of the placebo group, had detectable serum rIFNbeta levels. A small prospective time-course study was carried out in four MS patients receiving rIFNbeta. Serial blood samples were obtained prior to and 4, 8, 24, and 48 hours after rIFNbeta injection. A peak serum rIFNbeta level was observed between 8 and 24 hours after rIFNbeta injection and tended to decline to near preinjection levels at 48 hours postinjection. These results are consistent with the rationale of alternate-day, subcutaneous administration of rIFN beta. In addition, the ELISA described might be a useful tool to study the pharmacokinetics and the relationship of rIFN beta serum levels to clinical efficacy.

Differential up-regulation of HLA class I molecules on neuronal and glial cell lines by virus infection correlates with differential induction of IFN-beta.

Adult neurons normally lack the expression of MHC class I molecules, which has implications on virus clearance from the central nervous system. The author previously demonstrated that HLA class I up-regulation in measles virus (MV)-infected glial cells is primarily mediated by IFN-beta. In contrast, this study demonstrates that MV-infection of the neuronal cell lines IMR-32 and CHP-126 fails to up-regulate HLA class I expression, which was associated with an inability of MV to induce IFN-beta in the neuronal cell lines. However, treatment with IFN-beta on coculture of the IMR-32 neuronal cell line with MV-infected glioma cells resulted in the up-regulation of HLA class I on the former, which could be neutralized by anti-IFN-beta Ab. The inability of MV to up-regulate HLA class I expression on the neuronal cell line IMR-32 was not virus specific because similar findings were observed with mumps virus or stimulation with the synthetic dsRNA polyinosinic polycytidylic acid (PIPC). Induction of IFN-beta gene expression by virus requires binding of NF-kappa B to the positive regulatory domain II element of the IFN-beta promoter. Our studies indicate that MV, TNF-alpha, or PIPC induces NF-kappa B (p50 and p65 subunits) binding to positive regulatory domain II in the glioma cell line. In contrast, such activity was induced by TNF-alpha but not MV or PIPC in the neuronal cell line IMR-32. This indicated that HLA class I expression is differentially regulated in glial and neuronal cell lines in response to MV, which correlates with differential binding of NF-kappa B to the IFN-beta promoter and induction of IFN-beta gene expression.

Direct evidence that interferon-beta mediates enhanced HLA-class I expression in measles virus-infected cells.

Viral infection results in enhancement of HLA-class I expression in a number of cell types, including glial cells, which normally do not express these molecules. This enhancement may occur through a direct interaction between a viral component and the HLA-class I gene or indirectly through virus-induced soluble factors produced by infected cells. These include cytokines such as IFN-gamma, IFN-alpha/beta, and TNF-alpha, known to enhance class I expression. Measles virus (MV) infection of a human glioma cell line (U-105 MG) and of primary human umbilical vein endothelial cells enhances the expression of HLA-class I molecules on these cells. The enhancement of HLA-class I is dependent on infectious virus, as antibody-neutralized MV has no effect on class I expression. In this study, we demonstrate the presence of an HLA-class I-enhancing factor in supernatants from MV-infected cells. The supernatant class I-enhancing factor is not IFN-gamma, IFN-alpha, or TNF-alpha because MV-infected cells did not produce measurable levels of these cytokines as detected by immunoassay or polymerase chain reaction. In contrast, IFN-beta is produced by the infected cells and the supernatant class I-enhancing factor could be entirely neutralized by antibodies to IFN-beta, but not antibodies to IFN-alpha, TNF-alpha, or non-immune sera. Furthermore, preincubation of cells with neutralizing antibodies to IFN-beta prior to infection blocked MV enhancement of HLA-class I completely in the U-105 MG cells and by as much as 74% in the umbilical vein endothelial cells. The results of these experiments provide direct evidence that enhanced HLA-class I expression in MV-infected cells is mediated primarily by IFN-beta.

Different virus antibodies in serum and cerebrospinal fluid of patients suffering from subacute sclerosing panencephalitis.

Complement fixing (CF) antibody titers to measles, parainfluenza (PI) types 1 and 3, mumps, herpes type 1, and cytomegalovirus (CMV) in serum and cerebrospinal fluid (CSF) of 33 patients with subacute sclerosing panencephalitis (SSPE) were evaluated. Results were analyzed in comparison to 11 patients with neurological diseases other than SSPE and 7 normal subjects. All SSPE patients had elevated serum and CSF measles antibody titers. The number of SSPE patients manifesting elevated titers other than measles did not reach statistical significance when compared to controls, except for PI type 1. This suggests a possible dual infection with measles and PI in SSPE. The anticomplementary effect detected in the serum and CSF of some patients indirectly suggests the presence of immune complexes.

Cellular and humoral immunity in subacute sclerosing panencephalitis.

Cellular and humoral immunity was studied in 26 patients with subacute sclerosing panencephalitis. Results were compared with those of 14 normal controls and 11 patients suffering from other neurological disorders. It was shown that cellular and humoral immune responses are adequate in subacute sclerosing panencephalitis. The persistently elevated levels of serum immunoglobulin G (IgG) and IgA indicated a persistent infection, and their progressive rise in later stages correlated with the progressive nature of the illness. IgG progressively increased with the clinical stage in the cerebrospinal fluid unaccompanied by a corresponding rise in the measles antibody titer. This suggests that antigenic determinants other than those tested play a role in the production of IgG in the cerebrospinal fluid. The progressive increase in the ratio of cerebrospinal fluid to serum IgG with the advance of the disease suggests synthesis of IgG locally in the central nervous system. Elevated measles antibody titer in serum and cerebrospinal fluid is a consistent aid in the diagnosis of subacute sclerosing panencephalitis. It is more specific in cerebrospinal fluid than in serum. Its level did not vary significantly with the clinical stages or duration of illness. Depressed serum complement activity has been detected in some subacute sclerosing panencephalitis patients in whom serum levels of the third and fourth components of the complement were normal.