Mouse Protection Assay Research Articles

Boosting the Anti-Helicobacter Efficacy of Azithromycin through Natural Compounds: Insights From InVitro, InVivo, Histopathological, and Molecular Docking Investigations.

Antimicrobial-resistant Helicobacter pylori (H. pylori) poses a significant public health concern, especially given the limited therapeutic options for azithromycin-resistant strains. Hence, there is a necessity for new studies to reconsider the use of azithromycin, which has diminished in effectiveness against numerous strains. Thus, we aimed to augment azithromycin's anti-Helicobacter properties by combining it with curcumin in different formulations, including curcumin in clove oil, curcumin nano-gold emulsion, and curcumin nanoemulsion. The antimicrobial activities of the investigated compounds, both individually and in combination with other anti-Helicobacter drugs, were evaluated. Their antibiofilm and anti-virulence properties were assessed using both phenotypic and genotypic methods, alongside molecular docking studies. Our findings were further validated through mouse protection assays and histopathological analysis. We observed high anti-Helicobacter activities of curcumin, especially curcumin nanoemulsion. A synergistic effect was detected between curcumin nanoemulsion and azithromycin with fraction inhibitory concentration index (FICI) values <0.5. The curcumin nanoemulsion was the most active anti-biofilm and anti-virulence compound among the examined substances. The biofilm-correlated virulence genes (babA and hopQ) and ureA genes were downregulated (fold change <1) post-treatment with curcumin nanoemulsion. On the protein level, the anti-virulence activities of curcumin nanoemulsion were documented based on molecular docking studies. These findings aligned with histopathological scoring of challenge mice, affirming the superior efficacy of curcumin nanoemulsion/azithromycin combination. The anti-Helicobacter activities of all curcumin physical forms pose significant challenges due to their higher minimum inhibitory concentration (MIC) values exceeding the maximum permissible level. However, using curcumin nanoemulsion at sub-MIC levels could enhance the anti-Helicobacter activity of azithromycin and exhibit anti-virulence properties, thereby improving patient outcomes and addressing resistant pathogens. Therefore, more extensive studies are necessary to assess the safety of incorporating curcumin nanoemulsion into H. pylori treatment.

Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi.

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 μg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of β-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Paeoniflorin combined with norfloxacin ameliorates drug-resistant Streptococcus suis infection.

The increased resistance of bacterial pathogens to fluoroquinolones (FQs), such as norfloxacin and ciprofloxacin, supports the need to develop new antibacterial drugs and combination therapies using conventional antibiotics. The LuxS/AI-2 quorum sensing (QS) system can regulate the complex group behaviour of Streptococcus suis and impact its susceptibility to FQs. We investigated the combination of paeoniflorin and norfloxacin as a novel and effective strategy against FQ-resistant S. suis. FIC, AI-2 activity assay, real-time RT-PCR and biofilm inhibition assays were performed to investigate the in vitro effect of paeoniflorin combined with norfloxacin. Mouse protection and mouse anti-infection assays were performed to investigate the in vivo effect of paeoniflorin combined with norfloxacin. FIC results showed that paeoniflorin and norfloxacin exert a synergistic bactericidal effect. Evidence was brought that paeoniflorin reduces the S. suis AI-2 activity and significantly down-regulates the transcription of the FQ efflux pump gene. In addition, paeoniflorin can inhibit biofilm formation, thereby promoting the ability of norfloxacin to kill S. suis. Finally, we showed in a mouse model that paeoniflorin in association with norfloxacin is effective to treat S. suis infections. This study highlighted the inhibitory potential of paeoniflorin on the LuxS/AI-2 QS system of S. suis, and provided evidence that it can inhibit the FQ efflux pump and prevent biofilm formation to cooperate with norfloxacin in the treatment of resistant S. suis-related infections.

34920 Clinical immunogenicity of daxibotulinumtoxinA for injection (DAXI) in glabellar lines including subjects with multiple exposures: Pooled data from the SAKURA phase 3 trials

DAXI is a novel botulinum toxin type A (BoNTA) containing purified 150-kD core neurotoxin (daxibotulinumtoxinA) and proprietary stabilizing peptide (RTP004). As with any biologic, it was important to assess DAXI’s immunogenic potential. Neutralizing antibodies to daxibotulinumtoxinA and binding antibodies to daxibotulinumtoxinA or RTP004 were assessed in three phase 3 glabellar line studies (two placebo-controlled, single-dose studies [SAKURA 1 and 2] and an open-label study [SAKURA 3] of ≤3 treatments of 40U DAXI). Binding antibodies were detected by validated direct-binding ELISA in a multi-tiered (screening, confirmatory, and titration) assay. Subjects positive for daxibotulinumtoxinA-binding antibodies were evaluated for neutralizing antibodies (mouse protection assay). Overall, 2786 subjects received DAXI (n = 882, ≥2 treatments) and 2823 subjects were exposed to RTP004 as DAXI (n = 2786) or placebo (n = 203) (n = 914, ≥2 exposures). Of these, 2737 and 2772 were evaluable for daxibotulinumtoxinA and RTP004 antibodies, respectively. At baseline, 12/2737 (0.4%) subjects had binding antibodies to BoNTA and 66/2772 subjects (2.4%) had detectable RTP004 antibody titers. Treatment-emergent daxibotulinumtoxinA-binding antibodies were detected in 20 subjects (0.7%), and treatment-boosted binding antibodies in 1 subject (<0.1%). No subject developed daxibotulinumtoxinA-neutralizing antibodies. Treatment-induced anti-RTP004 binding antibodies were detected in 35 (1.3%) subjects. In most subjects, binding antibodies were transient. No subject had binding antibodies to both daxibotulinumtoxinA and RTP004. Transient binding antibodies to daxibotulinumtoxinA or RTP004 were found to not impact clinical efficacy, safety, or duration of action. No subjects developed neutralizing antibodies to daxibotulinumtoxinA. The findings suggest that DAXI administration results in a low incidence of antibody formation.

Antibody Development in Patients Treated Long-Term With OnabotulinumtoxinA for Benign Essential Blepharospasm and Hemifacial Spasm.

Report the development of onabotulinumtoxinA neutralizing antibodies in patients treated consecutively for 20 years or longer for benign essential blepharospasm (BEB), hemifacial spasm (HFS), and Meige Syndrome. Prospective, randomized, cross-sectional study of 12 randomly selected patients from a single clinical practice that have been treated consecutively for 20 or more years with onabotulinumtoxinA for BEB, HFS, or Meige Syndrome. Serum samples were collected from each subject and analyzed for neutralizing antibody formation using the Mouse Protection Assay. None of the tested patients (0%) displayed neutralizing antibodies to onabotulinumtoxinA. The mean duration of treatment was 27.5 years (range 22.1-34.1, SD 3.1, 95% confidence interval 25.45-29.50). Nine of the patients had a diagnosis of BEB, 2 HFS, and one Meige. Eleven of the 12 patients were women. There was no statistically significant difference in treatment dosage or interval over the course of treatment. The data support previous studies showing low incidence of antibody formation for botulinum A toxins with this subset of long-term treated patients. The results also provide further evidence for studies that have suggested increased onabotulinumtoxinA treatment volumes and/or decreased intervals between treatments are not due to neutralizing antibody formation and secondary non-response, but rather study designs that do not consider the titration phase of initial treatments. This study is specific to long-term treated patients, and the results cannot be generalized to patients naive to treatment.

Production and characterization of a neutralizing antibody against botulinum neurotoxin A

As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.

Editors' note: High prevalence of neutralizing antibodies after long-term botulinum neurotoxin therapy

In the article “High prevalence of neutralizing antibodies after long-term botulinum neurotoxin therapy,” Albrecht et al. investigated the prevalence of neutralizing antibodies (NAbs) against botulinum neurotoxin type A (BoNT/A) in a monocentric, observational cross-sectional study of 596 outpatients treated with BoNT/A over a mean of 5.3 years for different indications and found that 13.9% had measurable NAbs. The probability of developing NAbs increased with the single and cumulative dose of treatment and was associated with the BoNT/A formulation used, leading the authors to recommend avoiding booster injections and reducing the individual injected doses. In response, Dr. Jankovic notes that the authors did not mention other studies that showed a much lower frequency of NAbs, although after shorter periods of observation, and indicates that no data were provided regarding any correlation between the presence of NAbs and clinical response. In this regard, Dr. Jankovic notes that none of the patients in a previous study with blocking antibodies identified by the mouse protection assay (MPA) had any clinical response, suggesting that the MPA may be more clinically meaningful. In their reply, Albrecht et al. postulate that lower rates of NAbs in previous studies may relate to lower sensitivity of the MPA, lower BoNT doses used, and substantially shorter duration of treatment. In contrast to Dr. Jankovic's findings, they report that all their patients with NAbs opted to continue treatment and reported ongoing subjective benefit. Both the authors and Dr. Jankovic agree on the need for novel assays with higher sensitivity and specificity that are (ideally) not animal based. In another response, Mulroy et al. caution that the authors' conclusions may mislead readers into thinking that long-term treatment with frequent or repeated BoNT injections can result in clinical treatment failure. Like Dr. Jankovic, they too highlight the absence of data on clinical effectiveness in this study and note that NAb serostatus did not always correlate with clinical response in previous studies. They also seek to clarify the authors' disclosures, given that the study was funded by Merz Pharmaceuticals, which manufacturers one of the BoNT formulations studied. Replying to these comments, Albrecht et al. argue that there is consensus that the induction of NAbs should be avoided, as they are associated with reduced treatment response in several studies. They acknowledge that they do not have information about NAb induction with treatment intervals shorter than 10 weeks in their study, note the difficulty of comparing treatment efficacy across different indications, and concede that efficacy data were not available for all patients. They also state that pharmaceutical funding had no influence on the interpretation or analysis of their data. This correspondence highlights enduring uncertainty in the field regarding the clinical implications of NAbs as detected by existing animal-based assays. In the special article “Comprehensive systematic review summary: Disease-modifying therapies for adults with multiple sclerosis,” Rae-Grant et al. reported the findings of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology based on reviewing the evidence on starting, switching, and stopping disease-modifying therapies for clinically isolated syndrome, relapsing-remitting MS, and progressive forms of MS. In response, Dr. Tran, on behalf of Genentech, argues that the report did not make it clear that data on annualized relapse rate reduction and relative risk of in-study disability progression for ocrelizumab (presented along with other therapies in figures 1 and 3) were from direct comparisons with subcutaneous interferon-β-1a 44 μg 3 times weekly in 2 phase 3 randomized, double-blind, double-dummy trials. In response, Dr. Rae-Grant agrees that this information should have been included, but notes that statements in the text of the summary and in a key accompanying table acknowledged the superior efficacy of ocrelizumab compared with interferon- β-1a 3 times weekly. Dr. Rae-Grant also notes that data on subgroup analysis of treatment of highly active MS with ocrelizumab were not available at the time of publication of the systematic review.

RBC Adherence of Immune Complexes Containing Botulinum Toxin Improves Neutralization and Macrophage Uptake.

In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that the BoNT-neutralizing potency of mAbs can be improved through red blood cell (RBC) immunoadherence. For example, a fusion protein (FP) that adheres biotinylated mAbs to the RBC surface enabled a pair of mAbs to neutralize 5000 LD50 BoNT/A in the mouse protection assay. Here, we added two mAbs to that combination, creating a 4-mAb:FP complex that neutralized 40,000 LD50 BoNT/A in vivo, and analyzed functional correlates of neutralization. The FP enhanced potency of BoNT/A immune complexes, providing the greatest magnitude of benefit to the 4-mAb combination. RBC binding of a BoNT/A complexed with 4-mAb:FP exhibited a bi-phasic clearance process in vivo. Most of the complexes were cleared within five minutes; the rest were cleared gradually over many hours. Peritoneal macrophages showed better uptake of the 4-mAb complex than the 3-mAb complex, and this was not affected by the presence of the FP. However, the addition of RBCs to the 4-mAb:FP BoNT/A doubled macrophage uptake of the complexes. Lastly, the 4-mAb:FP BoNT/A complex synergistically induced M2 macrophage polarization, as indicated by IL-10 expression, whether or not RBCs were present. RBC-targeted immunoadherence through the FP is a potent enhancer of mAb-mediated BoNT/A neutralization in vivo, and can have positive effects on BoNT/A sequestration, immune complex uptake, and macrophage activation.

P.3.024 - Cardiovascular reactions to psychosocial stress and abuse history in patients with mood and anxiety disorders

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.

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

We have conducted a 26-month-long comparative study involving young patients (2–6years 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.

RNA aptamer as potential antidote against botulism: an in vivo report

Botulinum neurotoxins (BoNTs) are the most toxic substances to mankind, and yet, there are no therapeutics available to reverse the paralysis, the deadly symptom of botulism caused by BoNTs. Here we report that RNA aptamer, selected through in vitro process against light chain of botulinum neurotoxin type A (BoNT/A) showed in vivo protection efficacy against botulism. Our results showed that RNA aptamer not only accelerated the recovery of local paralysis induced by BoNT/A through mouse exercise wheel assay, but also significantly delayed the time-to-death through mouse protection assay, and rescued one mouse (out of six) completely intoxicated by BoNT/A. Our results demonstrated the potential using RNA aptamer as antidote against deadly botulism.

An enzyme-linked immunosorbent assay for detection of botulinum toxin-antibodies.

Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. We present an ELISA for BNT-AB detection that can be-for the first time-customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way.

An enzyme-linked immunosorbent assay for detection of botulinum toxin antibodies

Introduction Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. Methods Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. Results We present an ELISA for BNT-AB detection that can be—for the first time—customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. Conclusions Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way. © 2014 International Parkinson and Movement Disorder Society

Cross neutralisation of Southeast Asian cobra and krait venoms by Indian polyvalent antivenoms

Cross neutralisation of venoms by antivenom raised against closely-related species has been well documented. The spectrum of paraspecific protection of antivenom raised against Asiatic Naja and Bungarus (krait) venoms, however, has not been fully investigated. In this study, we examined the cross neutralisation of venoms from common Southeast Asian cobras and kraits by two widely used polyvalent antivenoms produced in India: Vins Polyvalent Antivenom (VPAV) and Bharat Polyvalent Antivenom (BPAV), using both in vitro and in vivo mouse protection assays. BPAV was only moderately effective against venoms of N. kaouthia (Thailand) and N. sumatrana, and either very weakly effective or totally ineffective against the other cobra and krait venoms. VPAV, on the other hand, neutralised effectively all the Southeast Asian Naja venoms tested, as well as N. naja, B. candidus and Ophiophagus hannah venoms, but the potency ranges from effective to weakly effective. In an in vivo rodent model, VPAV also neutralised the lethality of venoms from Asiatic Naja and B. candidus. In anesthetised rat studies, both antivenoms effectively protected against the N. kaouthia venom-induced cardio-respiratory depressant and neuromuscular blocking effects. Overall, our results suggest that VPAV could be used as alternative antivenom for the treatment of elapid envenomation in Southeast Asian regions including Malaysia, Thailand and certain regions of Indonesia.

Immunoresistance in Cervical Dystonia Patients After Treatment With AbobotulinumtoxinA

ABSTRACTFormation of antibodies against botulinum toxin type A has been observed following treatment of Cervical Dystonia (CD). We present the immunological findings from two 12-week Phase III prospective, randomized, double-blind, single-dose, placebo-controlled studies (Study 1, n = 116; Study 2, n = 136). Patients in both studies were administered abobotulinumtoxinA 500U or placebo intramuscularly at baseline. Patients could receive up to three or four additional treatments (250–1000U) in an open-label follow-up period. Blood samples were collected at baseline and during treatment to test for antibodies to abobotulinumtoxinA using a radioimmunoprecipitation assay (Study 2 only) and a mouse protection assay. Loss of response was predefined using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) total score at 4 weeks following injection. No subjects in Study 1 and one individual in Study 2 developed neutralizing antibodies (nABs) during the double-blind treatment phase; the individual who developed immunoresistance had received botulinum toxin type A treatment prior to the study and did not respond to treatment. Two subjects demonstrated a change in nAB status during open-label treatment and overall responsiveness was maintained in these patients. In conclusion, the development of immunoresistance was rare and, in the presence of circulating nABs, patients may still gain benefit from intramuscular abobotulinumtoxinA treatment.

Development of a humanized antibody with high therapeutic potential against dengue virus type 2.

BackgroundDengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.Methodology/Principal FindingsWe generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.Conclusions/SignificanceWe used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.

Meta‐analysis of neutralizing antibody conversion with onabotulinumtoxinA (BOTOX®) across multiple indications

This meta-analysis evaluated the frequency of neutralizing antibody (nAb) conversion with onabotulinumtoxinA (BOTOX®; Allergan) across five studied indications. The analysis was based on large, controlled or prospective, open-label trials (durations 4 months to ≥2 years). Serum samples were analyzed for nAbs using the Mouse Protection Assay. Subjects who were antibody negative at baseline and had at least one analyzable postbaseline antibody assay result were included. The 16 clinical studies included 3,006 subjects; of these, 2,240 met the inclusion criteria for this analysis. Subjects received 1-15 treatments (mean 3.8 treatments) with onabotulinumtoxinA. Total doses per treatment cycle ranged from 10 or 20 units in glabellar lines to 20-500 units in cervical dystonia. The numbers of subjects who converted from an antibody-negative status at baseline to antibody-positive status at any post-treatment time point were: cervical dystonia 4/312 (1.28%), glabellar lines 2/718 (0.28%), overactive bladder 0/22 (0%), post-stroke spasticity 1/317 (0.32%), and primary axillary hyperhidrosis 4/871 (0.46%). Across all indications, 11/2,240 subjects (0.49%) converted from antibody negative at baseline to positive at one or more post-treatment time points, but only three subjects became clinically unresponsive to onabotulinumtoxinA at some point following a positive assay. Based on these large trials, the frequency of antibody conversion after onabotulinumtoxinA treatment is very low, and infrequently leads to loss of efficacy. © 2010 Movement Disorder Society.

An Evaluation of Neutralizing Antibody Induction During Treatment of Glabellar Lines with a New US Formulation of Botulinum Neurotoxin Type A

The induction of neutralizing antibodies during the aesthetic application of botulinum neurotoxin type A is rare, but of potential clinical concern. Phase III studies of a new US formulation of botulinum neurotoxin type A, Dysport (BoNTA-ABO [abobotulinumtoxinA]; Medicis Aesthetics, Scottsdale, AZ), have not identified any cases of neutralizing antibody formation during the treatment of glabellar lines in patients who received up to nine treatments. To provide an in-depth analysis of the potential for induction of neutralizing antibodies in the study population enrolled in phase III trials of BoNTA-ABO in the treatment of glabellar lines. First and last available serum samples from patients in the BoNTA-ABO Glabellar Lines Development Program were screened for BoNTA-ABO antibodies with a radioimmunoprecipitation assay (RIPA), followed by a confirmatory competitive assay (RIPA-C). Confirmed RIPA-C-positive samples were further evaluated for the presence of neutralizing antibodies using a mouse protection assay (MPA), a highly specific bioassay for neutralizing antibodies. We conducted safety and efficacy evaluations, including day 30 responder rate (a rating of no or mild glabellar lines) and duration of response in the last treatment cycle. Of 1554 patients who received at least one BoNTA-ABO treatment (10 units at five injection points, for a total dose of 50 units/treatment; range one to nine treatments), five (0.32%) were antibody positive on the RIPA-C assay-two at baseline and three at the last treatment cycle. None of the RIPA-C-positive samples tested positive for neutralizing antibodies upon further evaluation using the highly specific MPA. Of note, the RIPA-C-positive group had a responder rate of 100% and a mean response of 103.3 days, while the RIPA-C-negative group had a responder rate of 90% and a mean response of 89.4 days. The safety of BoNTA-ABO did not appear to be altered in the RIPA-C-positive group. At the dose and treatment interval used in the correction of glabellar lines, induction of neutralizing antibodies to BoNTA-ABO was not observed. None of the five samples that initially gave positive results in a RIPA-C assay were positive when further evaluated using the MPA. Clinically, RIPA-C-positive status did not correlate with any reduction in efficacy or an altered safety profile, although the small numbers prevent definitive conclusions. These data suggest that the five RIPA-C-positive samples represented false positives.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Long‐term treatment with botulinum toxin type A in cervical dystonia has low immunogenicity by mouse protection assay

To evaluate the immunogenicity of botulinum toxin type A (BoNTA; BOTOX) in cervical dystonia (CD). Subjects diagnosed with CD for > or =1 year and previously naïve to BoNTs were treated with BoNTA in a prospective, open-label, multicenter study. Serum samples were analyzed for BoNTA neutralizing antibodies using the Mouse Protection Assay (MPA). Clinical resistance was assessed with a test injection of 20 U BoNTA placed unilaterally into the frontalis (Frontalis Antibody Test; FTAT) or corrugator muscle (Unilateral Brow Injection; UBI). Efficacy was assessed and adverse events were recorded. Of 326 subjects enrolled, 251 (77%) completed the study. Subjects received a median of 9 BoNTA treatments (mean dose per session ranged from 148.4 to 213.0 U over a mean of 2.5 years [range: 3.2 months-4.2 years]). Only 4 of 326 subjects (1.2%) tested positive for antibodies in the MPA; three of these subjects stopped responding clinically to BoNTA (of whom one also showed clinical resistance in the FTAT) and one continued to respond. Consistent improvements in the signs/symptoms of CD were noted. The most frequent treatment-related adverse events were mild to moderate weakness, dysphagia, neck pain, and injection-site pain. The current formulation of BoNTA rarely causes neutralizing antibody formation in CD subjects treated < or =4 years.