Antibody responses to botulinum neurotoxin type A of toxin-treated spastic equinus children with cerebral palsy: A randomized clinical trial comparing two injection schedules

Highlights

• Studied Ab response link to HLA in toxin-naïve spastic equines children with cerebral palsy treated with BoNT/A tri-annually or annually.

• Blocking Abs detected in 2 out of 18 sera samples in the tri-annual group but none in 20 samples from the annual group.

• The blocking Abs positive sera samples gave in RIA higher anti-BoNT/A Ab-binding levels than the negative samples.

• Higher Ab binding in RIA correlating with blocking Abs in two samples in the tri-annual group.

• By combining data from CD studies, association between DQB1*06:04 and DQA1*01:02 and Ab-positivity was significant.

Abstract

We have conducted a 26-month-long comparative study involving young patients (2–6 years old) with a clinical diagnosis of spastic equinus secondary to cerebral palsy who have been treated with BoNT/A (BOTOX®, Allergan) tri-annually or annually. Serum samples were obtained to determine the presence or absence of blocking antibodies (Abs) by a mouse protection assay (MPA) and levels of anti-BoNT/A Abs by radioimmune assay (RIA). HLA DQ alleles were typed using blood samples to determine the possible association of certain HLA type(s) with the disease or with the Ab status. Blocking Abs were detected in only two out of 18 serum samples of the tri-annual group, but none were found in 20 samples of the annual group. The MPA-positive serum samples gave in RIA significantly higher anti-BoNT/A Ab-binding levels than the MPA-negative samples. On the other hand, when two MPA-positive sample data were excluded, serum samples from tri-annual and annual groups showed similar anti-BoNT/A Ab levels. Linkage of the disorder with a particular HLA DQA1 and DQB1 allele types was not observed due to the small sample size. However, by combining results with other studies on BoNT/A-treated Caucasian patients with cervical dystonia (CD), we found that, among Caucasian patients treated with BoNT/A, DQA1*01:02 and DQB1*06:04 were higher in Ab-positive than in Ab-negative patients. The genetic linkage was on the threshold of corrected significance.

Introduction

Botulinum neurotoxins (BoNTs) (MW, 150 kDa) are a group of most potent protein neurotoxins produced by Clostridium botulinum (Lamanna, 1959). Currently eight BoNT serotypes (types A through H) are known. They exert toxicity by blocking acetylcholine release at the nerve terminals in the neuromuscular junction (Simpson, 1989, Schiavo et al., 1992). The neuro-paralyzing activity of BoNTs is reversible and therefore injections in minute doses, especially of type A (BoNT/A) and type B (BoNT/B), have been applied clinically for a variety of disorders (Aoki, 2002, Jankovic, 2004). Governmental approval of BoNT use varies by country, but in the US, the FDA approved BoNT/A as a therapeutic agent in 1989 for a limited number of disorders (Jankovic and Brin, 1997), and for CD in 2000. Many indications have since been approved. Further, an increasing number of off-label use of BoNT/A in various treatments have been reported (Brin, 2009). The treatments require repeated injections at 3–6-month intervals, which might cause some patients to develop unwanted blocking antibody (Ab) responses that cause unresponsiveness (Jankovic, 2002, Jankovic, 2006, Jankovic, 2009, Atassi, 2004, Brin et al., 2008). But the production of blocking Abs that interfere with treatment has become a low (1–2%) incidence since the current form of BOTOX® was introduced (Jankovic et al., 2003, Jankovic et al., 2006, Jankovic, 2006, Jankovic, 2009, Brin et al., 2008, Naumann et al., 2010).

Spastic equinus is a condition characterized by toe walking on one or both feet and the most common underlying diagnosis is children with spastic cerebral palsy (CP). BoNT/A injections have been used for treatment and management of spastic equinus in cerebral palsy for more than two decades (Koman et al., 1993, Cosgrove et al., 1994, Graham et al., 2000, Gibson et al., 2007, Naidu et al., 2010, Love et al., 2010). The first large randomized controlled study evaluating the efficacy and safety of BoNT/A in children with CP (n = 114; age, 2–16 years) was a short-term trial with observation ending at 12 weeks (Koman et al., 2000). Long-term studies using BoNT/A on CP have been rare until a 12–56 month long (mean 33 months) study was conducted, in which BoNT/A was injected at 3-month interval with a fixed dose for young children (n = 8; age, 4.7 ± 2 years) with equinus gait (Garcia Ruiz et al., 2000). Many other studies on proper selection of doses, injection intervals or administration sites (Satila et al., 2006, Satila et al., 2008) have followed. However the effect on the treatment of BoNT/A in a strict control of dose and frequencies of administration has not been well studied. Moreover, apart from a few reports which described the detection of blocking Abs in these treatments (Koman et al., 2001, Kanovsky et al., 2009), how treatment protocol for spastic equinus affects the immunological responses against BoNT/A has not been investigated. Therefore, in the present work, we have carried out a randomized clinical trial (RCT) comparing two frequencies of BOTOX® injections for the management of spastic equinus in toxin-naïve children with CP as well as their immunological assessment. One group (n = 18) received BOTOX® every four months (total of 6 injections) and the other (n = 20) every 12 months (total of 2 injections), over a 26-month period. The BOTOX® dose per kilogram body weight was standardized in both groups. The presence of blocking Abs and levels of Abs against BoNT/A were determined and compared between two groups. We also determined HLA DQ haplotypes of each patient to examine if association between certain HLA types and spastic equinus as well as other immunological status can be found. We report here the immunological results of this study. Clinical results comparing the two injection schedules have been reported previously (Hastings-Ison et al., 2016).