Botulinum neurotoxins (BoNTs), produced by the anaerobic spore-forming bacterium Clostridium botulinum, are among the most potent known biological toxins. BoNTs cause lethal botulism via contaminated food, wound infections, or infant intestinal colonization, posing significant threats to public health. Although the mouse bioassay is still being considered as the gold standard for detecting BoNTs, its drawbacks, including the lengthy experimental duration, high costs, and ethical issues, highlight the urgent need to develop alternative methods to fulfill the detection requirements. In recent years, frequent botulism poisoning incidents haves put forward higher requirements for detection technology. On-site detection is expected to be rapid and immediate, while laboratory detection requires high sensitivity and serotype discrimination capabilities. This review comprehensively introduces current detection approaches, including mouse bioassay, cell-based assays, immunological methods, endopeptidase–mass spectrometry, biosensors, chromatography, and mass spectrometry techniques. Notably, cell-based assays have been used for the potency testing of commercialized botulinum toxin type A and are considered the most promising alternative to the mouse bioassay. Biosensors based on nanomaterials demonstrate advantages in real-time detection due to their rapid response and portability, while endopeptidase–mass spectrometry achieves high sensitivity and effective serotype identification by specifically recognizing toxin-cleaved substrates. Future works shall aim to completely replace MBA, developing a detection system suitable for multiple scenarios such as clinical diagnosis, food safety monitoring, and environmental monitoring. The detection methods should also have matrix compatibility and serotype discrimination capabilities.

The study was to evaluate the safety, immunogenicity and lot-to-lot consistency of live attenuated varicella vaccine in Chinese population aged 1–3 years. The double-blind, randomized phase III trial was conducted in Henan Province, China. In total, 1197 subjects were included in this study. Subjects were randomly assigned into four groups in a 2:2:2:1 ratio to receive one of the three lots of commercial scale (CS) vaccine or the licensed pilot scale (LPS) vaccine. Seroconversion rate and neutralizing antibody titers (NATb) were assessed at day 0 pre-vaccination and at day 30 post-vaccination. Safety data were recorded for 30 days post-vaccination. After vaccination, the geometric mean titers (GMTs) of the three CS groups were 25.04 (95% confidence interval [CI], 22.85 to 27.44), 24.47 (95% CI, 22.35 to 26.78) and 25.88 (95% CI, 23.61 to 28.36), respectively (P = 0.6928). The ratio of GMTs adjusted for covariates of each pair of lots were all between 0.67 to 1.50 in susceptible subjects. The difference of seroconversion rate between pooled CS group and LPS group was 3.82 (95% CI, 0.55 to 8.81). Meanwhile, the percentage of solicited local, systemic and unsolicited adverse reactions showed no difference across the four groups, and most of the adverse reactions were mild or moderate in intensity. The CS group was comparable to the LPS group in safety and immunogenicity. The consistency of three consecutive CS lots was reliable. Moreover, the CS group was non-inferior to the LPS group.

(1) Background: At present, the only potency assay approved in China for the in-country testing of botulinum toxin type A for injection products is the mouse bioassay (MBA). The Chinese market for neurotoxin products is rapidly expanding, but MBAs are subject to high variability due to individual variations in mice, as well as variations in injection sites, in addition to the limited number of batches tested for one MBA. Compared with the mLD₅₀ method, the cell-based potency assay (CBPA) developed for the potency testing of onabotulinumtoxinA (BOTOX) by AbbVie not only does not use any experimental animals but also allows for significant time and cost savings. Due to the significant benefits conferred by the replacement of the mLD₅₀ assay with CBPA in China, the CBPA method has been transferred, validated, and cross-validated to demonstrate the equivalence of the two potency methods. (2) Methods: The differentiated SiMa cells were treated with both BOTOX samples and the reference standard, and the cleaved SNAP25₁₉₇ in the cell lysates was quantified using Chemi-ECL ELISA. A 4-PL model was used for the data fit and sample relative potency calculation. The method accuracy, linearity, repeatability, and intermediate precision were determined within the range of 50% to 200% of the labeled claim. A statistical equivalence of the two potency methods (CBPA and mLD₅₀) was initially demonstrated by comparing the AbbVie CBPA data with NIFDC mLD₅₀ data on a total of 167 commercial BOTOX lots (85 50U lots and 82 100U lots). In addition, six lots of onabotulinumtoxinA (three 50U and three 100U) were re-tested as cross-validation by these two methods for equivalence. (3) Results: The overall assay’s accuracy and intermediate precision were determined as 104% and 9.2%, and the slope, R-square, and Y-intercept for linearity were determined as 1.071, 0.998, and 0.036, respectively. The repeatability was determined as 6.9%. The range with the acceptable criteria of accuracy, linearity, and precision was demonstrated as 50% to 200% of the labeled claim. The 95% equivalence statistic test using margins [80%, 125%] indicates that CBPA and mLD₅₀ methods are equivalent for both BOTOX strengths (i.e., 50U and 100U). The relative potency data from cross-validation were within the range of ≥80% to ≤120%. (4) Conclusions: The CBPA meets all acceptance criteria and is equivalent to mLD₅₀. The replacement of mLD₅₀ with CBPA is well justified in terms of ensuring safety and efficacy, as well as for animal benefits.

Pertussis remains a leading cause of infant mortality globally, with rising cases in China post-COVID-19. Despite vaccination, waning immunity from acellular pertussis vaccines has driven resurgence. We evaluated a novel diphtheria, tetanus and pertussis (acellular, three components) combined vaccine, adsorbed (DTcP), China's first genetically engineered three-component pertussis vaccine, administered under two primary schedules (2/3/4 vs. 2/4/6 months) compared to licensed DTaP-IPV-Hib (Pentaxim). In this randomized, blinded, phase III clinical trial, 1380 healthy 2-month-old infants from Henan, China, received DTcP-1 (2/3/4 months) or DTcP-2 (2/4/6 months) or DTaP-IPV-Hib (2/3/4 months). The primary safety endpoints were the incidence of adverse reactions within 0∼30 days after primary vaccination. The primary immunogenicity endpoints were to evaluate the non-inferiority and superiority of seroconversion rates and geometric mean concentrations (GMCs) of anti-pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), tetanus toxoid (TT), antibodies 30 days after primary vaccination. Immunogenicity was assessed via Luminex-based multiplex immunoassay. Both DTcP-1 and DTcP-2 schedules demonstrated non-inferior safety to DTaP-IPV-Hib (total adverse reactions: 14.52/16.59% vs. 16.91%, P = 0.183), with DTcP-2 (2/4/6 months) showing lower swelling (2.59% vs. 4.83%, P = 0.008) and irritability (0.07% vs. 1.02/1.40%, P < 0.001). DTcP-2 elicited higher GMCs against pertussis antigens (PT: 84.23 vs. 65.35; FHA: 132.16 vs. 102.13, both P < 0.001) and comparable DT/TT responses. DTcP exhibited favorable safety and superior pertussis immunogenicity, particularly with the 2/4/6-month schedule. Its genetically engineered three-component design offers a promising strategy to combat pertussis amid global resurgence.