Christian Ross

Clinical importance of neutralising antibodies against interferon beta in patients with relapsing-remitting multiple sclerosis.

BACKGROUND Interferon beta is the first-line treatment for relapsing-remitting multiple sclerosis, but the drug can induce neutralising antibodies against itself, which might reduce effectiveness. We aimed to assess the clinical effect of neutralising antibodies. METHODS We measured neutralising antibodies every 12 months for up to 60 months in 541 patients with multiple sclerosis, randomly selected from all patients who started treatment with interferon beta between 1996 and 1999. Patients left the study if they changed or discontinued therapy. Antibodies were measured blindly, using antiviral neutralisation bioassays with high, medium, and low sensitivity, and with different neutralising capacities as cutoff value for definition of a neutralising-antibody-positive result. FINDINGS Patients developed neutralising antibodies independent of age, sex, disease duration, and progression index at start of treatment. Relapse rates were significantly higher during antibody-positive periods (0.64-0.70) than they were during antibody-negative periods (0.43-0.46; p<0.03). When comparing the number of relapses in the neutralising-antibody-positive and neutralising-antibody-negative periods we found odds ratios in the range 1.51 to 1.58 (p<0.03). Time to first relapse was significantly increased by 244 days in patients who were antibody-negative at 12 months (log rank test 6.83, p=0.009). During this short-term study, presence of neutralising antibodies did not affect disease progression measured with the expanded disability status scale. INTERPRETATION Our findings suggest that the presence of neutralising antibodies against interferon beta reduces the clinical effect of the drug. In patients who are not doing well on interferon beta, the presence of such antibodies should prompt consideration about change of treatment.

Immunogenicity of interferon-β in multiple sclerosis patients: Influence of preparation, dosage, dose frequency, and route of administration

A total of 754 consecutive patients with relapsing‐remitting multiple sclerosis were investigated for interferon‐β (IFNβ) antibodies by protein‐G affinity chromatography and antiviral neutralization bioassay during 24 months on 6 MIU (22 μg) of subcutaneous IFNβ‐1a once weekly (n = 143) or three times weekly (n = 160), 6 MIU (30 μg) of intramuscular IFNβ‐1a once weekly (n = 140), or 8 MIU every other day of IFNβ‐1b (n = 311). The proportion of binding antibodies was higher in those receiving IFNβ‐1b compared with 6 MIU of IFNβ‐1a three times weekly (97 vs 89% at 12 months), and fewer became positive if 6 MIU of IFNβ‐1a was administered once weekly (58 vs 89%). Fewer patients on intramuscular than subcutaneous IFNβ‐1a became positive (33 vs 58%). The binding and neutralizing capacities were higher in the IFNβ‐1b group than in the IFNβ‐1a groups; these differences, however, were not significant after 12 months. The number of positive patients varied considerably and depended on the amount of IFN added to the bioassay; adding 10 LU/ml or more masked antibody detection. Antibodies induced by either preparation neutralized both IFNβ species but not IFNα. In conclusion, IFNβ‐induced antibodies are frequently found in multiple sclerosis patients, and IFNβ‐1b is more immunogenic than IFNβ‐1a. The immunogenicity of IFNβ‐1a increases with the frequency of administration and if it is given subcutaneously. Ann Neurol 2000;48:706–712

Appearance and disappearance of neutralizing antibodies during interferon-beta therapy

Background: Neutralizing antibodies (NABs) occur frequently in patients receiving interferon (IFN)-beta for multiple sclerosis (MS), but it is unclear whether occurrence of NABs is predictive for the persistence of NABs during continued IFN-beta therapy. Methods: The authors used an antiviral neutralization bioassay to measure NABs blindly from 6 months up to 78 months in patients with MS who were followed for at least 24 months during treatment with IFN-beta. Patients were classified into three groups: 1) persistently NAB-negative patients, defined as patients without any positive samples at any time; 2) definitely NAB-positive patients, defined as patients who had at least two consecutive positive samples; and 3) patients with fluctuating NAB-positive and NAB-negative samples. Results: A total of 455 patients were included in the study. Overall, 52.3% of the patients were persistently NAB-negative, 40.9% became definitely NAB-positive, and the remaining 6.8% were fluctuating. More patients treated with IFN-beta-1a (Avonex) remained NAB-negative (p < 0.0001), whereas there was no difference between IFN-beta-1b (Betaferon) and IFN-beta-1a (Rebif). Patients who have remained NAB-negative during the first 24 months of therapy rarely developed NABs. On the contrary, the majority of patients, who had been NAB-positive from 12 through 30 months after start of therapy, remained NAB-positive. Conclusions: NABs should be measured in all patients treated with IFN-beta. If patients have been persistently NAB-negative for 24 months, measurements can be discontinued. Patients who have been NAB-positive for a period of 18 months or more usually remain NAB-positive for a long time.

High-avidity autoantibodies to cytokines

Abstract The increased use of recombinant cytokines and cytokine analogues in various disease therapies emphasizes the importance of antibodies to cytokines. Here, Klaus Bendtzen and colleagues discuss how immunological tolerance to cytokines may be broken, and the clinical relevance of cytokine autoantibodies. They also comment on the implications for refined passive immunization and cytokine vaccination strategies.

Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN‐alpha 2b and human IFN‐gamma in healthy blood donors

Recently, naturally occurring antibodies to IFN‐α were discovered in a few systemic lupus erythematosus (SLE) and cancer patients; however, in most patients monitored for anti‐IFN antibodies before treatment, no antibodies were found. In an attempt to explain the ‘IFN‐blocking effect’ that we observed in all serum samples we investigated 200 sera from healthy blood donors. We isolated the globulin fraction, and used rabbit anti‐human IgG and IgM columns, protein A columns and T‐gel affinity chromatography to isolate human IgG and IgM. All sample fractions were tested in a biological IFN neutralization assay by means of a sensitive MTT‐assay. We found that normal human serum contained autoantibodies to crude human leucocyte IFN, native human fibroblast IFN, recombinant human leucocyte IFN‐α2b and recombinant human IFN‐γ, and that these naturally occurring antibodies were biologically active immunoglobulins of IgG and IgM type. These anti‐IFN antibodies were also present in purified human normal immunoglobulin pools. We conclude that all humans have naturally occurring anti‐interferon antibodies in their serum, and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by immunoglobulins.

Neutralizing antibodies hamper IFNβ bioactivity and treatment effect on MRI in patients with MS

We measured neutralizing antibodies (NABs) and the in vivo biologic response to interferon-β on neopterin and β2-microglobulin blood levels. All NAB-negative patients had an in vivo biologic response (full or partial), whereas all high-level positive patients had no response. High-level NAB patients had more MRI activity than NAB-negative patients (p = 0.031). Patients with a full response had less MRI activity than patients without biologic response (p = 0.032).

Persistence of neutralizing antibodies after discontinuation of IFNβ therapy in patients with relapsing-remitting multiple sclerosis

Objective The main objective was to follow serum levels of neutralizing antibodies (NABs) against interferon-beta (IFNβ) after discontinuation of IFNβ therapy. Background A large proportion of patients treated with recombinant IFNβ for multiple sclerosis (MS) develop therapy-induced NABs. Knowledge of persistence of NABs after discontinuation of therapy is limited. Design/patients: A retrospective follow-up study of patients treated in Denmark for relapsing-remitting (RR) MS with IFNβ for at least 12 months. NAB-positive patients, who discontinued therapy, were followed up with measurements of NABs. Methods We measured NAB-neutralizing capacity and NAB titres a.m. Kawade using a clinically validated cytopathic effect assay. Results Thirty-seven patients were included. Mean follow-up time was 22 months. Of the 29 patients with a NAB titre at or above 25 prior to termination of therapy, only three patients reverted to a titre below 25. Of these, two had a titre below 200 and one patient a titre of 600 at the last examination before treatment stop. The longest post-treatment follow-up during which a patient maintained NAB positivity was 59 months. Conclusion NABs against IFNβ, especially with high titres, tend to persist for a long time after discontinuation of IFNβ therapy. NABs should always be measured before reinstitution of IFNβ treatment in NAB-positive patients.

Measuring and evaluating interferon beta-induced antibodies in patients with multiple sclerosis

Author for correspondence: Klaus Bendtzen, Institute for Inflammation Research IIR 7521, Rigshospitalet University Hospital, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. E-mail: address: kben@mail.dk Administration of interferons (IFNs) may induce antibodies that interfere with therapeutic efficacy. We have optimized and validated methods for large-scale economic screening. Sera from patients with relapsing-remitting multiple sclerosis (MS) were investigated for binding antibody (BAb) by protein-G affinity-chromatography radioimmunoassay and a commercially available enzyme immunoassay (EIA). Neutralizing antibody (NAb) was investigated by cytopathic effect assays (CEA) using both fixed amount and serially diluted sera. BAb correlated with log10-transformed titres obtained by EIA (r=0.70, p<0.0001); the latter, however, failed to demonstrate low-level BAb. Comparison of clinical significance of NAb-positivity measured by biological assays with different sensitivities demonstrated an optimal odds ratio for relapse rate using 10 laboratory units (LU)/mL. Purification of IgG prior to CEA removed toxicity from toxic sera. The neutralizing capacity data correlated linearly with log10-transformed titres obtained by a Kawade 10-to-1 LU/mL CEA (r=0.77, p<0.0001). In conclusion, neutralizing capacity CEA utilizing a fixed amount of serum predicts differences in relapse rates in IFNβ-treated MS patients and correlates with NAb titres of the 10-to- 1 LU/mL CEA. Neutralizing capacity CEA is less laborious and more economical than titre-based NAb assays and suitable for large-scale screenings of MS patients.

Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii

Background An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. Methods P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Results Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). Conclusion We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

A sensitive antiviral neutralization bioassay for measuring antibodies to interferons

An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low OD signals. This fall in OD can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the OD signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte IFN although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques.