Assessment of Key Elements in the Innate Immunity System Among Patients with HIV, HCV, and Coinfections of HIV/HCV.

BACKGROUND: Coinfection of Hepatitis C virus (HCV) with human immunodeficiency virus (HIV) has a higher risk of mortality than HCV or HIV monoinfection. HCV and HIV infections are specified by systemic inflammation, but the inflammation process in HCV/HIV coinfection is much complicated and is not well characterized. OBJECTIVE: The aim of this study was to analyze the expression of TLR-3, TLR-7, IL-10, IFN-1 (IFN-α, IFN-ß), and TNF-α in HIV, HCV and HIV/HCV co-infected patients. METHODS: Forty-five patients including HIV group (n=15), HCV group (n=15), HIV/HCV coinfection group (n=15) and healthy control group (n=15) participated. Peripheral blood mononuclear cells (PBMCs) were obtained. PBMC-RNA, HCV and HIV RNA were extracted from all subjects and cDNA was synthesized. The viral load analyzed by reverse transcription-quantitative PCR (RT-qPCR), and the expression levels of IFN-α, IFN-ß, TLR-3, TLR-7, TNF, and IL-10 mRNA were quantified in PBMCs. RESULTS: The levels of IFN-I, IL-10, and TNF-α were overexpressed in all patients' groups (p<0.05), TLR-7 was upregulated in all groups, but this upregulation was not statistically significant (p>0.05). TLR-3 showed a decrease in all patient groups (p<0.05). The statistical analysis demonstrated that TLR-3 has a negative correlation with HIV load, whereas other genes positively correlated with HIV load. In addition, TLR-3, TNF-α, and IFN-I were negatively correlated with HCV load, whereas TLR-7 and IL-10 s were positively correlated with HCV load. CONCLUSION: Our results showed a significant relationship between the expression level of innate immunity genes and inflammation in HCV, HIV, and HIV/HCV coinfected patients.

In vitro assessment of the biologic activity of interferon beta formulations used for the treatment of relapsing multiple sclerosis.

A new formulation (NF) of subcutaneous (sc) interferon (IFN) ß-1a was developed in an attempt to improve injection tolerability and immunogenicity. We compared antiviral and IFNß-stimulated gene (ISG) activities of IFNß-1a sc NF with IFNß-1a sc original formulation and IFNß-1b sc. When equivalent unit amounts were compared, the IFNß formulations demonstrated similar antiviral activity and induced similar levels of ISG mRNA. However, on a weight basis (ng/mL), significantly more IFNß-1b sc was needed to equal the antiviral activity of either IFNß-1a sc formulation, and both IFNß-1a sc formulations induced significantly higher levels of ISG mRNA than IFNß-1b sc.

Socio-economic aspects of the testing for antibodies in MS-patients under interferon therapy in Austria: a cost of illness study.

BACKGROUND: According to EU-guidelines testing patients on interferon-beta (IFNb) for the presence of neutralising antibodies (NAb) is recommended; IFNb treatment efficacy of NAb-positive patients equals that of placebo-treated patients. Economic impact of NAb testing in MS patients has not been explored yet. The aim of this analysis is to estimate the impact of NAb testing in RRMS-patients on Austria׳s health-care-system. METHODS: A decision-analytic model over 5 years was performed. The cost effectiveness of NAb testing versus no testing was evaluated. The model considers switching after relapse and withdrawal. All direct costs are based on Austrian data from 2013 and were discounted at 5% per year. The efficacy outcome measure was "relapse free". Clinical data and resource use were determined by literature. RESULTS: Total costs for all Austrian MS-patients on IFNb-therapy with testing amount to 187,554,021€ over 5 years; without testing is 175,091,300 €. Costs per relapse avoided over 5 years were 90,075€ in the NAb testing arm, and 99,535€ in the no NAb test arm, resulting in a difference of 9460€ in favour of routine NAb testing. Considering all 3590 IFNb-treated patients 2082 relapses can be avoided in the NAb testing arm versus 1759 in the no-testing arm within 5 years. Testing for NAb leads to costs per relapse avoided of 18,015€ per year versus 19,907€ when no tests are done. CONCLUSION: The results suggest that NAb testing reduces relapses and associated costs.

Guidelines on the clinical use for the detection of neutralizing antibodies (NAbs) to IFN beta in multiple sclerosis therapy: report from the Italian Multiple Sclerosis Study group.

Interferon beta (IFNß) was the first specific disease-modifying treatment licensed for relapsing-remitting multiple sclerosis, and is still one of the most commonly prescribed treatments. A strong body of evidence supports the effectiveness of IFNß preparations in reducing the annual relapse rate, magnetic resonance (MRI) disease activity and disease progression. However, the development of binding/neutralizing antibodies (BAbs/NAbs) during treatment negatively affects clinical and MRI outcomes. Therefore, guidelines for the clinical use for the detection of NAbs in MS may result in better treatment of these patients. In October 2012, a panel of Italian neurologists from 17 MS clinics convened in Milan to review and discuss data on NAbs and their clinical relevance in the treatment of MS. In this paper, we report the panel's recommendations for the use of IFNß Nabs detection in the early identification of IFNß non-responsiveness and the management of patients on IFNß treatment in Italy, according to a model of therapeutically appropriate care.

Use of a standardized MxA protein measurement-based assay for validation of assays for the assessment of neutralizing antibodies against interferon-ß.

Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-ß in multiple sclerosis (MS) patients on IFN-ß therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-ß products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-ß products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-ß NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-ß1a rather than IFN-ß1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-ß1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described.

An assessment of biological potency and molecular characteristics of different innovator and noninnovator interferon-beta products.

Approved innovator products and their noninnovator "copy" versions are likely to vary in their quality, eg, physicochemical characteristics and biological activity, with important implications for clinical efficacy and safety. Therefore, it is important to study and thoroughly evaluate the noninnovator products in comparison with approved products at the preclinical and clinical stages. We have obtained 4 noninnovator interferon (IFN)-ß-1a products currently marketed in Latin America and Iran and compared these with approved IFN-ß-1a products (Avonex and Rebif) obtained from the same geographical regions with respect to biological potency, estimated by in vitro bioassays, and molecular characteristics, assessed by immunoblotting and high-performance liquid chromatography. In this article, we present our data showing that the noninnovator IFN-ß-1a products can vary considerably in their biological potency. In addition, we showed that all IFN-ß-1a products formulated with human serum albumin contained variable amounts of higher-molecular-weight aggregates of IFN-ß-1a and adducts with human serum albumin, these being more prevalent in 2 noninnovator IFN-ß-1a products where biological potency was reduced compared with approved IFN-ß-1a products. Additionally, significant lot-to-lot variability was observed for one of the noninnovator products. Taken together, the results of this study highlight the need for not only thorough in vitro characterization, but also preclinical and clinical assessment to ensure patient safety and efficacy.

Critical review: assessment of interferon-ß immunogenicity in multiple sclerosis.

This review discusses type I interferon (IFN) immunogenicity with focus on methods of detection of anti-IFN antibodies in patients treated with human recombinant IFN-β. Pitfalls involved in the clinical use of various types of assays for binding antibodies and neutralizing antibodies against IFN-β are presented, and the widely held distinction between binding antibodies and neutralizing antibodies is questioned both in terms of detection and clinical importance. The article also addresses important bioavailability and pharmacokinetic issues occurring with prolonged use of protein drugs. The rationale for individualized or personalized medicine, ie, optimizing therapies according to individual needs rather than using standardized trial-and-error regimens to all patients, is highlighted.

Evaluation of a multiplex bead-based screening assay for detection of binding antibodies to interferon-beta.

Multiple sclerosis patients treated with interferon-beta (IFNbeta) can develop neutralizing binding antibodies (BAbs) that reduce the agent's effectiveness. Screening for these antibodies can be performed by ELISA. We investigated a multianalyte immune detection (MAID) assay as an alternative to ELISA to detect anti-IFNbeta-1a and -1b. For 146 sera representing both 1a and 1b treated groups, MAID concordance with ELISA was 94% and 92%, respectively. For all discordant results, the corresponding ELISA and MAID values were within 4 units of each other, and all discordant values but one fell within 2 units of the BAbs cutoff value for reflexing to neutralization testing (4 units). Our data indicate that the MAID assay is an accurate and cost-effective alternative to ELISA for detecting BAbs to IFNbeta.

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.

Neutralizing antibodies to interferon beta: assessment of their clinical and radiographic impact: an evidence report: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology.

The clinical and radiologic impact of developing neutralizing antibodies (NAbs) to interferon beta (IFNbeta) while on this therapy for multiple sclerosis (MS) is assessed. On the basis of Class II and III evidence, it is concluded that treatment of patients with MS with IFNbeta (Avonex, Betaseron, or Rebif) is associated with the production of NAbs (Level A). NAbs in the serum are probably associated with a reduction in the radiographic and clinical effectiveness of IFNbeta treatment (Level B). In addition, the rate of NAb production is probably less with IFNbeta-1a treatment than with IFNbeta-1b treatment, although the magnitude and persistence of this difference is difficult to determine (Level B). Finally, it is probable that there is a difference in seroprevalence due to variability in the dose of IFNbeta injected or in the frequency or route of its administration (Level B). Regardless of the explanation, it seems clear that IFNbeta-1a (as it is currently formulated for IM injection) is less immunogenic than the current IFNbeta preparations (either IFNbeta-1a or IFNbeta-1b) given multiple times per week subcutaneously (Level A). However, because NAbs disappear in some patients even with continued IFNbeta treatment (especially in patients with low titers), the persistence of this difference is difficult to determine (Level B). Although the finding of sustained high-titer NAbs (>100 to 200 NU/mL) is associated with a reduction in the therapeutic effects of IFNbeta on radiographic and clinical measures of MS disease activity, there is insufficient information on the utilization of NAb testing to provide specific recommendations regarding when to test, which test to use, how many tests are necessary, or which cutoff titer to apply (Level U).

Interferon beta-1b (betaseron/betaferon) is well tolerated at a dose of 500 microg: interferon dose escalation assessment of safety (IDEAS).

The approved interferon beta-1b (Betaseron/Betaferon) dose is 250 microg (8 MIU) administered subcutaneously (sc) every other day (eod). Clinical trial data suggest a dose response effect for interferon beta in multiple sclerosis (MS) treatment and a maximum dose has yet to be established. The Interferon Dose Escalation Assessment of Safety (IDEAS) study evaluated the safety and tolerability of interferon beta-1b 500 microg (16 MIU) sc eod with structured dose escalation and adverse event (AE) management in 22 patients (20 interferon beta-1b-treated (SD) and two interferon beta-1b-naïve (ND)) with relapsing-remitting (RR) MS, secondary-progressive (SP) MS, or progressive relapsing MS. IDEAS comprised an eight-week dose escalation period and a 12-week maintenance period, with modification as clinically warranted. Autoinjectors were used for all injections > or =0.4 mL. Clinical laboratory values were monitored monthly. Baseline and exit assessments included the MS Functional Composite score, EDSS, and neutralizing antibody MxA assay. AEs were recorded at every injection. Dose escalation ranged from two to 12 weeks. Some 91% of patients (20/22) achieved the 500-microg dose, and of these 90% (18/20) completed the maintenance phase. There were no differences in response between ND and SD patients. Most common AEs were decreased general well-being, insomnia, and injection site reactions (mostly mild). The 500-microg dose of interferon beta-1b was well tolerated in the short-term with escalation and premedication in these patients, most of whom had previously been receiving 250 microg interferon beta-1b.

Benefit-risk assessment of interferon-beta therapy for relapsing multiple sclerosis.

IFN-beta therapy has a central place in the management of relapsing multiple sclerosis, as demonstrated by the pivotal studies of three IFN-beta treatment regimens. However, questions remain concerning the optimal choice of preparation and dose regimen. The benefit-risk ratio for a given preparation is an important consideration in optimising treatment for an individual patient. Of the three IFN-beta preparations currently available, all have shown benefit on activity measures (relapses and active lesions apparent on magnetic resonance imaging), while benefit on progression measures (disability and total lesion burden) has been less consistent. Available data across studies suggest that dose and/or dose frequency are important determinants of efficacy, a finding supported by direct comparative data on different IFN-beta preparations. The added benefit of high-dose, high-frequency IFN-beta therapy is not achieved at the cost of compromised safety or tolerability, indicating that three-times-weekly treatment offers a superior benefit-risk ratio to once-weekly treatment. The potential advantages of IFN-beta therapy may be enhanced by regular monitoring of efficacy and safety in order to maintain patients on therapy beyond the first few months when side effects are most apparent.

Assessment and management of neutralizing antibodies in patients with multiple sclerosis.

The development of neutralizing antibodies (NAbs) to interferon beta (IFNbeta) products during treatment of MS poses a challenge for clinicians. Given the impact of NAbs on the clinical efficacy of IFNbetas, the immunogenicity of different IFNbeta products should be one of the factors that neurologists consider in the treatment of patients with MS. However, no clear guidelines are available for the practicing neurologist concerning which patients should be tested for NAbs and how to clinically manage patients who develop NAbs. This article summarizes the content of this supplement, discusses issues related to measuring NAbs in clinical practice, and gives practical alternatives for managing MS in patients who develop NAbs during IFNbeta therapy.

Interferon-beta-1b: a review of its use in relapsing-remitting and secondary progressive multiple sclerosis.

Interferon-beta-1b (Betaseron, Betaferon) is a non-glycosylated recombinant human interferon-beta approved for high-frequency, subcutaneous (SC) administration in the treatment of multiple sclerosis (MS). Its mechanism of action is unknown, but may involve modulation of the autoimmune pathogenic processes of MS. In a randomised, double-blind trial in patients with relapsing-remitting MS (RRMS), SC interferon-beta-1b 250 micro g (8 million International Units [MIU]) every other day reduced the annual relapse rate and increased the proportion of relapse-free patients compared with placebo. It also reduced relapse severity, hospitalisations, and disease activity assessed by magnetic resonance imaging (MRI), and increased the time to first relapse. Progression of disability showed a trend towards reduction relative to placebo and baseline, but did not reach statistical significance. SC interferon-beta-1b 250 micro g every other day was shown in a randomised trial to be superior to intramuscular (IM) interferon-beta-1a 30 micro g (6 MIU) once weekly with respect to reductions in relapse-related parameters, disability progression and MRI-assessed disease activity. In patients with secondary progressive MS (SPMS), SC interferon-beta-1b 250 micro g every other day slowed progression of the disease relative to placebo in one randomised, double-blind trial, but not in another. In both studies, interferon-beta-1b 250 micro g recipients had fewer relapses and less MRI-assessed disease activity than placebo recipients. The difference in primary outcome may reflect differences in patient entry criteria. Interferon-beta-1b is generally well tolerated and the common adverse events (e.g. injection site reactions, asthenia and an influenza-like symptom complex) are clinically manageable. In a randomised trial, the tolerability of SC interferon-beta-1b 250 micro g every other day was generally similar to that of IM interferon-beta-1a 30 micro g once weekly, except for higher incidences of injection site reactions and neutralising anti-interferon-beta antibodies with SC interferon-beta-1b. In conclusion, SC interferon-beta-1b 250 micro g every other day reduces the frequency and severity of relapses and MRI measures of disease activity and may delay the progression of disability in RRMS. The drug appeared to be more effective than, and as well tolerated as, IM interferon-beta-1a 30 micro g once weekly. Interferon-beta-1b also has positive effects on relapse rates and disease activity in patients with SPMS, although its effects on disease progression remain uncertain. The drug is generally well tolerated, and the common adverse events are clinically manageable. Thus, interferon-beta-1b is a valuable first-line therapy for patients with RRMS and a potentially useful option in those with SPMS.

A sensitive radioimmunoprecipitation assay for assessing the clinical relevance of antibodies to IFN beta.

BACKGROUND: Some multiple sclerosis (MS) patients treated with interferon beta (IFN beta) develop antibodies to the drug. Neutralising antibody (NAB) assays for IFN beta are expensive and the clinical relevance of the results has been debated. OBJECTIVE: To establish a cheap, sensitive, and reliable assay for antibodies to (125)I-IFN beta, and to correlate levels of antibodies with clinical response to IFN beta treatment. METHODS: We established a radioimmunoprecipitation assay (RIPA) using (125)I-IFN beta. We tested NAB positive sera, healthy control sera, and serial samples of 33 IFN beta-1b treated MS patients from the Vancouver cohort of the Berlex pivotal trial who had a high incidence of NABs. RESULTS: We found that the RIPA was highly sensitive for the detection of antibodies to IFN beta-1a and -1b, and that there was a strong correlation between reactivity of NAB positive sera for (125)I-IFN beta-1b and for (125)I-IFN beta-1a. The RIPA was more sensitive and consistent than the NAB. Moreover, there was a trend towards poorer MRI outcomes in RIPA positive patients, but not in NAB-positive patients. CONCLUSIONS: The RIPA assay is sensitive and easy to perform. It should be of value in assessing the clinical impact of IFN beta antibodies, and its use could help target expensive INF beta treatments to those who will respond best.

[The assessment of development of binding antibodies to interferon beta during interferon beta 1a treatment of multiple sclerosis]. / Ocena powstawania przeciwcial wiazacych interferon beta w trakcie leczenia stwardnienia rozsianego interferonem beta 1-a.

The importance of binding antibodies (BAb) that develop during the treatment of multiple sclerosis with interferon beta has not been fully explained yet. However, they are generally regarded as one of factors that may diminish treatment efficacy. The aim of the study was to evaluate firstly, BAb occurrence in interferon beta 1-a (IFN beta 1-a)-treated MS patients and secondly, BAb impact on clinical efficacy of this medication. In the 36-month study participants were 21 patients with relapsing-remitting multiple sclerosis, RR-MS, (14 women, 7 men, aged 29.6 +/- 8.5). All the patients were receiving intramuscular IFN beta-1a (Avonex) for 24 months, in the dose of 30 micrograms per week. Clinical parameters and serum BAb levels (the EIA method) were estimated every 3 months. Two control groups, examined only once, included 20 RR-MS patients without IFN-beta therapy and 20 healthy volunteers. While before treatment a high BAb level was found in 2 patients (9.5%), at 6 months of treatment it was found in 8 patients (38.1%). A similar number of patients with high BAb levels was seen throughout the study during the IFN-beta treatment. On therapy completion serum BAb levels decreased very rapidly. After 2 years of treatment, disability as measured by the EDSS scale was more pronounced in patients with serum BAb, but the differences were statistically not significant. No statistically significant relationship was found either between elevated BAb levels and the number of relapses during the IFN-beta treatment (including relapses that required steroid therapy).