Neutralizing antibodies explain the poor clinical response to interferon beta in a small proportion of patients with multiple sclerosis: a retrospective study.

BACKGROUND: Neutralizing antibodies (NAbs) against Interferon beta (IFNbeta) are reported to be associated with poor clinical response to therapy in multiple sclerosis (MS) patients. We aimed to quantify the contribution of NAbs to the sub-optimal response of IFNbeta treatment. METHODS: We studied the prevalence of NAbs in MS patients grouped according to their clinical response to IFNbeta during the treatment period. Patients were classified as: group A, developing >or= 1 relapse after the first 6 months of therapy; group B, exhibiting confirmed disability progression after the first 6 months of therapy, with or without superimposed relapses; group C, presenting a stable disease course during therapy. A cytopathic effect assay tested the presence of NAbs in a cohort of ambulatory MS patients treated with one of the available IFNbeta formulations for at least one year. NAbs positivity was defined as NAbs titre >or= 20 TRU. RESULTS: Seventeen patients (12.1%) were NAbs positive. NAbs positivity correlated with poorer clinical response (p < 0.04). As expected, the prevalence of NAbs was significantly lower in Group C (2.1%) than in Group A (17.0%) and Group B (17.0%). However, in the groups of patients with a poor clinical response (A, B), NAbs positivity was found only in a small proportion of patients. CONCLUSION: The majority of patients with poor clinical response are NAbs negative suggesting that NAbs explains only partially the sub-optimal response to IFNbeta.

Neutralizing and binding antibodies to interferon beta in patients with multiple sclerosis: a comparison of assay results from three italian centres.

Interferon (IFN) beta therapy for multiple sclerosis (MS) is associated with the development of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in a percentage of patients. This study investigated the reproducibility of results of two different antibody detection techniques using serum from 100 patients with MS who were receiving IFN beta therapy. Fifty samples were analysed using a commercially available kit-based BAb assay and a further 50 different samples were analysed using a widely used NAb cytopathic effect assay, at three different laboratories. All three centres agreed on the BAb status of all serum samples. However, only 84% agreement was reached on serum NAb status, and there was significant inter-laboratory variation in NAb titre values. Further analysis of these data revealed a correlation between the mean NAb titre and the coefficient of variation of serum samples, indicating greater discordance with higher NAb titres. A significant interlaboratory variation in NAb titres does exist; thus, caution is required when comparing titres from different centres. It is clear that validated detection assays are needed to accurately quantify NAb titres.

Immunogenicity comparison of interferon beta-1a preparations using the BALB/c mouse model: assessment of a new formulation for use in multiple sclerosis.

The in vivo immunogenicity of a new interferon (IFN) beta-1a product (Rebif New Formulation; RNF) was compared with that of two approved recombinant human IFN beta-1a products (Rebif and Avonex). Immunogenic potential was assessed based on time to development of neutralizing antibodies (NAbs) and NAb titer. Female BALB/c mice (six in each group) received RNF, Rebif or Avonex (1.0 microg/mL subcutaneously three times weekly), and serum samples collected on Days 7, 21, and 35 (Study 1), or 28, 42, 49, and 60 (Study 2) were assayed for NAbs. In Study 1, no mice had NAbs at Day 7, but by Day 21 one mouse in the RNF group had NAbs, compared with three and four mice in the Rebif and Avonex groups, respectively. Results were similar in Study 2. All control mice were NAb negative; all actively treated mice had NAbs by day 35 or 42. Throughout Study 1, NAb titers were lowest in the RNF group and highest in the Avonex group, and at day 35, NAb titers were significantly lower in the RNF group than the Rebif group (p = 0.037). Results indicate that, on a gram-for-gram basis, RNF appears less immunogenic than Rebif or Avonex.

Genotypic resistance of archived and circulating viral strains in the blood of treated HIV-infected individuals.

The aim of this study was to evaluate patterns of antiretroviral resistance of HIV-1 in peripheral blood mononuclear cells (PBMCs) and in the plasma of patients whose therapeutic regimen is failing. Plasma and PBMC samples were collected from 95 HIV-infected patients undergoing long-term treatment. Genotyping of the reverse transcriptase (RT) and protease genes of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Comparison of the amino acid sequence of the RT and protease genes in cell-associated variants of HIV-1 with that of the plasma revealed that 62 of the 95 patients' samples tested exhibited different genotypic resistance patterns (discordant samples [DSs]). In 27% of samples, the patterns of resistance detected were concordant in both compartments. In 51% of DSs, the greatest number of mutations was found in plasma; however, in 37% of DSs, greater numbers of mutations were found in PBMC DNA. The HIV mutation patterns detected in plasma do not necessarily reflect those found in the cell-associated compartment. The observation that the cellular compartment may contain an archive of the resistance variant makes this reservoir an interesting substrate for analysis of the "resistance potential" in a given patient.

The effects of prolonged treatment with zidovudine, lamivudine, and abacavir on a T-lymphoblastoid cell line.

A human T-lymphoblastoid cell line that is resistant to the antiviral activity of zidovudine (ZDV) and moderately resistant to lamivudine (3TC) has been obtained as a result of prolonged treatment with a combination of three nucleoside analogues (NA), ZDV, 3TC, and abacavir (ABV). These cells, called CEM(ZLA), are fully sensitive to ABV. The cellular resistance of the CEM(ZLA) cells to ZDV correlates with significant reductions in thymidine kinase (TK) activity and in the amount of intracellular TK protein. Interestingly, the reduction in TK activity led to impairment of the ability of CEM(ZLA) to accumulate the triphosphate metabolite of ZDV. However, the moderately 3TC-resistant phenotype of CEM(ZLA) cannot be ascribed to a similar reduction in deoxycytidine kinase activity. Compared to the parental CEM cells, CEM(ZLA) cells express a high level of multidrug resistance protein 4 (MRP4), which could reduce the intracellular concentration of 3TC. This study shows that the exposure of cells to a combination of NAs is capable of simultaneously affecting more than one target site to confer resistance and that NAs display differing abilities to select cellular resistance mechanisms.

A novel assay to detect low-titred antibodies to interferon beta in multiple sclerosis patients.

Neutralizing antibodies (NAbs) may compromise interferon (IFN) clinical efficacy in patients with multiple sclerosis (MS) receiving IFN-beta treatment. When bioassays are used for anti-IFN-beta antibody detection, they are unable to discriminate between NAbs or other interfering substances with anti-IFN activity. Here we report the development of an anti-IFN-beta Western blot method that facilitates the detection of IFN low-titred antibodies and characterizes such low neutralizing activity as specifically due to the presence of particular IFN antibodies. The assay was characterized using serum samples from patients with MS treated with IFN-beta. It was developed by adding anti-IFN-positive antibody sera to Dynabeads M-280 tosylactivated followed by Western blot analysis. All sera samples from MS patients with IFN-betala NAbs (< or = 50 t1/10) proved to be antibody-positive using this new method and, more importantly, four of 27 binding antibody-negative sera samples were scored as IFN antibody-positive. The method was found to be rapid, specific and sensitive and consistent with respect to well-established antiviral neutralization or commercial enzyme-linked immunosorbent assays.

Neutralizing antibodies against endogenous interferon in myasthenia gravis patients.

Anti-cytokine antibodies (Abs) play an important role in the regulation of the immune response, both under normal conditions and in several autoimmune and neoplastic disorders. In the present study, we have investigated the occurrence and the clinical significance of natural neutralizing Abs (NAbs) against interferons (IFNs) alpha, beta, and gamma, as detected by bioassay, in 52 patients with myasthenia gravis (MG), and 43 sex- and age-matched healthy individuals. Patients showing titres > or = 1.3 Log t(1/10), confirmed in 2 consecutive samples collected two months apart, were considered positive. NAbs against any of the IFNs were not detected in healthy subjects. Of the 52 MG patients, 11 (21.1%) had NAbs against IFNalpha and three (5.8%) had NAbs against IFNbeta. None of these patients was found to be positive for NAbs against IFNgamma. Of the patients positive for NAbs against IFNalpha, eight (15.4%) had NAbs at titres > or =2 Log t(1/10). A positive association was observed between high titres of NAbs and the presence of thymoma. These data suggest the presence of a generalized activation of the humoral response in MG.

Increased sensitivity of SARS-coronavirus to a combination of human type I and type II interferons.

There is currently an urgent need to identify effective antiviral agents that will prevent and treat severe acute respiratory syndrome coronavirus (SARS-CoV) infection. In this study, we have investigated and compared the antiviral effect of different interferons (IFNs) on SARS-CoV replication in the epithelial kidney monkey Vero cell line. The results showed that SARS-CoV grown in Vero cells is moderately sensitive to IFN-beta and only weakly sensitive to IFN-alpha and IFN-gamma, in comparison to other IFN-sensitive viruses, such as those for encephalomyocarditis, vesicular stomatitis and Newcastle disease. Simultaneous incubation of Vero cells with IFN-beta and IFN-gamma indicated that they may act synergistically against SARS-CoV replication. The IFN-induced MxA protein was detected in the IFN-treated Vero cells. The data, however, suggest that the antiviral activity of IFN against SARS-CoV virus is independent of MxA expression.

Fate of neutralizing and binding antibodies to IFN beta in MS patients treated with IFN beta for 6 years.

An increasing number of evidence is showing that during prolonged treatment of relapsing-remitting multiple sclerosis (RRMS) with interferon (IFN) beta 1a or IFN beta 1b, the patients may develop serum anti-IFN antibody. It has been argued that some of the RRMS patients receiving IFN beta, who developed antibodies to IFN, lose them over time even though the treatment continues. To gain further insights into this issue, we performed a study to establish what happened to binding antibodies (BAB) and neutralizing antibodies (NAB) in 42 RRMS patients treated for 6 years with IFN beta 1a and/or IFN beta 1b. While the data of BAB analysis did not allow to reach definite conclusions, the results on NAB development confirm that the presence of this type of antibodies is transitory; in fact, most of the positive patients reverted to seronegative, although the IFN treatment is still ongoing; the only patients who were positive for NAB at 6 years of treatment are those whose serum contains high concentration of them. The paper also shows that patients lose antibodies to IFN independently on the type of IFN used for the treatment. In conclusion, the data indicate that the disappearance of the anti-IFN antibodies from the serum while the patients are still undergoing IFN treatment depends on the titer of antibodies but not on the type of IFN administered.

Serum interferon (IFN)-neutralizing antibodies and bioactivities of IFNs in patients with severe type II essential mixed cryoglobulinemia.

The efficacy of alpha interferon (IFN-alpha) in the treatment of severe type II essential mixed cryoglobulinemia (EMC) has been reported previously. In some patients, the development of neutralizing antibodies to recombinant IFN-alpha (rIFN-alpha) can affect the clinical response achieved with rIFN-alpha; a second treatment with natural IFN-alpha preparations may reinduce the clinical response. In the present study the ability of leukocyte IFN (LeIFN) to restore the response was investigated from a pharmacodynamic viewpoint. Specifically, the pharmacodynamic profiles of different IFN-alpha preparations were studied by measuring the serum neopterin levels and the levels of expression of protein MxA mRNA in in vivo peripheral blood mononuclear cells in two patients with EMC whose resistance to rIFN-alpha2a treatment increased concomitantly with the development of neutralizing antibodies. These markers were measured before injection and at 24 and 48 h after a single injection of rIFN-alpha2a, consensus IFN [(C)IFN], or LeIFN. No increase or only a slight increase in MxA mRNA levels was detectable after administration of rIFN-alpha2a or (C)IFN, whereas a significant increase (>/=10-fold) in MxA mRNA expression was recorded following administration of LeIFN. The neutralizing antibodies to rIFN-alpha2a cross-react with (C)IFN. Sera from these patients neutralized most but not all of the subtypes present in the natural IFN-alpha (LeIFN) mixture, and no significant increase in neopterin levels was observed after these patients were switched to LeIFN treatment. In summary, the data demonstrate that the problem of neutralizing antibodies still exists and that LeIFN may induce an increase in the level of MxA mRNA expression but not an increase in neopterin levels in patients who are resistant to treatment with rIFN-alpha2a or (C)IFN.

Cellular issues relating to the resistance of HIV to antiretroviral agents.

It has been proposed that the declining efficiency of antiretroviral agents in human immunodeficiency virus (HIV) infection may also depend on cellular factors at their site of action. Two in particular have been proposed: (i) the defective intracellular metabolism of NRTI in target cells and the altered uptake; and (ii) efflux of nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) by cellular transporter molecules. Several studies have shown that: changes in the activities of various purine and pyrimidine biosynthetic enzymes may occur in lymphocytes of HIV-infected patients; HIV-infected patients on prolonged treatment with nucleoside analogues, e.g. zidovudine, show significantly decreased activity of thymidine kinase (TK) compared with untreated HIV-infected people; and NRTI and PI are substrates for the multidrug membrane transporters. With regard to the latter issue, it is known that the ATP-binding cassette transporter proteins such as the P-glycoprotein (MDR), and the newly discovered family of multidrug resistance-associated proteins (MRP1-6), promote the active extracellular efflux of a wide variety of therapeutics drugs and overexpression of some of them lowers intracellular concentration of PI. In the very near future such mechanisms, also called 'cellular drug resistance', might be taken into account, together with other immunological, virological and behavioural factors, to explain the 'drug failure' and/or the variability of response in HIV patients undergoing antiretroviral treatment.

Selection of a T-cell line resistant to stavudine and zidovudine by prolonged treatment with stavudine.

It has been demonstrated that prolonged treatment with nucleoside analogues, such as 3'-azido-3'-deoxythymidine (zidovudine), 2',3'-dideoxycytidine (zalcitabine) and 9-(2-phosphonylmethoxyethyl) adenine (PMEA), may cause selection of cells that are resistant to their anti-HIV activity. A human T-lymphoblastoid cell line that is resistant to the antiviral and cytotoxic activity of 2',3'-didehydro-3'-deoxythymidine (stavudine) has developed as a result of prolonged treatment. These cells, called CEMstavudine, are also less sensitive to zidovudine. The cellular/pharmacological resistance acquired by the CEMstavudine cells is relatively low and appears to correlate with a reduction in thymidine kinase (TK) activity, rather than with a decreased expression of TK mRNA.

Proteolytic balance in patients with multiple sclerosis during interferon treatment.

Multiple sclerosis (MS) is a progressive, inflammatory, demyelinating disease. An altered cytokine network has been reported to occur during the disease, and its pathogenetic role has been hypothesized. To date, interferon-beta (IFN-beta) is the most effective and reliable therapy in the majority of MS patients, although the mechanisms underlying its therapeutic effects are not fully understood. Breakdown of the blood-brain barrier (BBB) with consequent extravasation of the T cells and their invasion of the brain parenchyma seems to be one of the most important steps in the pathogenesis of the disease. Matrix metalloproteinease-2 (MMP-2) and MMP-9 are enzymes with proteolytic activities toward extracellular matrix ECM components. They are physiologically balanced by the MMP tissue inhibitors TIMP-2 and TIMP-1, so that proteolysis occurs as the result of increased MMP or decreased TIMP levels. In 38 patients with MS, MMP-2 and TIMP-1 levels were similar before and after 9 months of IFN-beta therapy, whereas MMP-9 levels significantly decreased and TIMP-2 levels significantly increased in comparison to values obtained before treatment. These results suggest that IFN-beta modulates T cell activities, including MMP and TIMP production, thus contributing either to maintaining the integrity of the BBB or to slowing the progression of the disease.

Neutralizing and binding antibodies to IFN-beta: relative frequency in relapsing-remitting multiple sclerosis patients treated with different IFN-beta preparations.

The frequencies of anti-interferon-beta (IFN-beta) antibody development reported to date in patients treated with different IFN-beta preparations are not readily comparable mainly because of differences in underlying diseases and assay methods. Thus, the frequency of neutralizing antibody (NAb) and binding antibody (BAb) development was analyzed in a sample of sera derived from a homogeneous group of relapsing-remitting multiple sclerosis (RRMS) patients treated with different IFN-beta preparations. The frequency of developing NAb and BAb to IFN-beta varied according to the IFN-beta given. Specifically, the NAb seroconversion frequency was significantly higher in patients treated with Betaferon, Schering AG, Berlin, Germany (31.3%) than in patients treated with both preparations of recombinant IFN-beta 1a (Rebif, Serono, Geneva, Switzerland [7.4%] or Avonex, Biogen, Cambridge, MA [6.3%]). Analysis of BAb seroconversion frequency in the same patients revealed that different IFN-beta preparations may also have different capability to induce BAb development and that BAb are produced during IFN-beta therapy at a significantly higher rate than NAb. Our main conclusion is that different human IFN-beta preparations may possess different immunogenicities, leading to varying frequency of development of antibody to IFN-beta in RRMS.