Botulinum and Tetanus Neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

The tyrosine phosphatases LAR and PTPRδ act as receptors of the nidogen-tetanus toxin complex.

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system.

A Brief History of Polyclonal Antibody Therapies Against Bacterial and Viral Diseases Before COVID-19.

The use of the serum or plasma of patients or animals who have recovered from an infectious disease, or had been immunized with a relevant antigen, to treat or prevent the same infection in others began in the late 1880s when French and German scientists uncovered, one step at a time, several of the elements of the immune system's response to infection. A key finding was that the damage caused by some bacteria depends upon their secreted toxins which can be neutralized by biologic agents. Antitoxins to diphtheria and tetanus began to be manufactured in large animals in France, Germany, and the US in the 1890s and were soon being used worldwide. The impact of diphtheria antitoxin on childhood mortality was profound. Shortly after the development of antitoxins, convalescent serum began to be used for its anti-bactericidal properties thus addressing serious infections caused by non-toxin-producing organisms. The effectiveness of antitoxins and antisera was demonstrated by examining mortality rates in hospitals before and after the introduction of antitoxins, by comparisons of treated and untreated patients, by comparing early and late treatment and dosage, by examining vital data mortality trends, and by several randomized and alternate assignment trials. Antitoxins continue to have a role in the rare cases of diphtheria and other conditions largely eradicated by immunization, but serum therapy nearly disappeared from the medical armamentarium with the development of antibiotics in the 1940s. Inasmuch as new human pathogens are now emerging with unprecedented regularity as seen in the recent COVID-19 pandemic, and because specific therapies are unlikely to be available for them, plasma-based antibody therapies are likely to again carve out a niche in infectious disease control.

Serologic Immunity to Tetanus in the United States, National Health and Nutrition Examination Survey, 2015-2016.

BACKGROUND: Tetanus, a life-threatening infection, has become rare in the United States since introduction of tetanus toxoid-containing vaccines (TTCVs), recommended as a childhood series followed by decennial boosters beginning at age 11-12 years; vaccination uptake is high in children but suboptimal in adults. The objective of this study was to estimate the prevalence of sero-immunity to tetanus among persons aged ≥6 years in the United States and to identify factors associated with tetanus sero-immunity. Understanding population protection against tetanus informs current and future vaccine recommendations. METHODS: Anti-tetanus toxoid antibody concentrations were measured for participants of the 2015-2016 National Health and Nutrition Examination Survey (NHANES) aged ≥6 years for whom surplus serum samples were available using a microsphere-based multiplex antibody capture assay. Prevalence of sero-immunity, defined as ≥0.10 IU/mL, was estimated overall and by demographic characteristics. Factors associated with tetanus sero-immunity were examined using multivariable regression. RESULTS: Overall, 93.8% of the US population aged ≥6 years had sero-protection against tetanus. Prevalence of sero-immunity was above 90% across racial/ethnic categories, sex, and poverty levels. By age, ≥ 90% had protective sero-immunity through age 69 years, but prevalence of sero-immunity declined thereafter, with 75.8% of those aged ≥80 years having protective sero-immunity. Older age (adjusted prevalence ratio [aPR]: 0.89, 95% confidence interval [CI]: .85-.92) and being born outside the United States (aPR: 0.96, 95% CI: .93-.98) were significantly associated with lower prevalence of sero-immunity. CONCLUSIONS: The majority of the US population has vaccine-induced sero-immunity to tetanus, demonstrating the success of the vaccination program.

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vß sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vß repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Clinical profile and outcomes of tetanus patients in a tertiary hospital in the Philippines: a ten-year retrospective study.

BACKGROUND: Tetanus is a life-threatening but preventable neurologic disorder characterized by trismus and muscle spasms. Despite its decreasing global incidence, it remains to be endemic in resource-limited settings such as the Philippines. This study aimed to determine the incidence, demographic characteristics, risk factors, clinical presentation, management, complications, and outcomes of non-neonatal tetanus cases in a tertiary hospital in the Philippines. It also aimed to compare the clinical profile and outcomes between the adult and pediatric subgroups. METHODS: This study used a retrospective cross-sectional design including all adult and pediatric non-neonatal tetanus patients admitted at the University of the Philippines - Philippine General Hospital from January 2012 to June 2023. Data was extracted from department censuses and inpatient charts. RESULTS: One hundred thirty-eight cases were included. The incidence rate was 0.03%, while mortality rate was 29%. Majority of patients were males presenting with trismus and spasms after sustaining a puncture wound. Chronic hypertension was associated with an increased hazard of death by 4.5 times (p = 0.004), while treatment with magnesium sulfate was associated with a decreased hazard of death by 35 times (p = 0.005). The mode of infection and the medications administered differed between the adult and pediatric subgroups. CONCLUSIONS: Although the total number of cases has decreased over the past decade, tetanus remains to have a high incidence and mortality rate in the Philippines. Increasing vaccination coverage, improving public awareness, and educating health professionals can help reduce morbidity and mortality from this disease.

Disruption of layer-specific visual processing in a model of focal neocortical epilepsy.

The epileptic brain is the result of a sequence of events transforming normal neuronal populations into hyperexcitable networks supporting recurrent seizure generation. These modifications are known to induce fundamental alterations of circuit function and, ultimately, of behavior. However, how hyperexcitability affects information processing in cortical sensory circuits is not yet fully understood. Here, we investigated interlaminar alterations in sensory processing of the visual cortex in a mouse model of focal epilepsy. We found three main circuit dynamics alterations in epileptic mice: (i) a spreading of visual contrast-driven gamma modulation across layers, (ii) an increase in firing rate that is layer-unspecific for excitatory units and localized in infragranular layers for inhibitory neurons, and (iii) a strong and contrast-dependent locking of firing units to network activity. Altogether, our data show that epileptic circuits display a functional disruption of layer-specific organization of visual sensory processing, which could account for visual dysfunction observed in epileptic subjects. Understanding these mechanisms paves the way to circuital therapeutic interventions for epilepsy.

Autopsy of tetanus with foot gangrene as portal of entry.

Non-traumatic chronic skin lesions are the second most common cause of tetanus. Herein, we describe an 85-year-old woman who presented with a chronically infected skin lesion. She developed tetanus while in hospital and died of respiratory failure, after refusing mechanical ventilation. Routine immunization against tetanus began in Japan during 1968; hence many people born before 1968 are unvaccinated. Mortality due to tetanus is high and the proportion with protective antibodies is low in older adults. Therefore, we recommend tetanus vaccination for older persons in Japan who have chronic skin lesions and have never been vaccinated.

Introduction of a hexavalent vaccine containing acellular pertussis into the national immunization program for infants in Peru: a cost-consequence analysis of vaccination coverage.

BACKGROUND: Infant vaccination coverage rates in Peru have declined in recent years, exacerbated by the COVID-19 pandemic. Introduction of the fully-liquid diphtheria, tetanus, and acellular pertussis (DTaP)-inactivated polio vaccine (IPV)-hepatitis B (HB)-Haemophilus influenzae type B (Hib) hexavalent vaccine (DTaP-IPV-HB-Hib) in Peru's infant National Immunization Program may help improve coverage. We evaluated costs and healthcare outcomes, including coverage, of switching from a pentavalent vaccine containing whole-cell pertussis component (DTwP-HB-Hib) plus IPV/oral polio vaccine (IPV/OPV) to the hexavalent vaccine for the primary vaccination scheme (2, 4 and 6 months). METHODS: The analysis was performed over a 5-year period on a cohort of children born in Peru in 2020 (N = 494,595). Four scenarios were considered: the pentavalent plus IPV/OPV scheme (S1); replacing the pentavalent plus IPV/OPV scheme with the hexavalent scheme (S2); expanded delivery of the pentavalent plus IPV/OPV scheme (S3); expanded delivery of the hexavalent scheme (S4). Vaccine coverage and incidence of adverse reactions (ARs) were estimated using Monte Carlo simulations and previous estimates from the literature. Cases of vaccine-preventable diseases were estimated using a Markov model. Logistical and healthcare costs associated with these outcomes were estimated. Impact of key variables (including coverage rates, incidence of ARs and vaccine prices) on costs was evaluated in sensitivity analyses. RESULTS: The overall cost from a public health payer perspective associated with the pentavalent plus IPV/OPV vaccine scheme (S1) was estimated at $56,719,350, increasing to $61,324,263 (+ 8.1%), $59,121,545 (+ 4.2%) and $64,872,734 (+ 14.4%) in scenarios S2, S3 and S4, respectively. Compared with the status quo (S1), coverage rates were estimated to increase by 3.1% points with expanded delivery alone, and by 9.4 and 14.3% points, if the hexavalent vaccine is deployed (S2 and S4, respectively). In both scenarios with the hexavalent vaccine (S2 and S4), pertussis cases would also be 5.7% and 8.7% lower, and AR rates would decrease by 32%. The cost per protected child would be reduced when the hexavalent vaccine scheme. Incidence of ARs was an important driver of cost variability in the sensitivity analysis. CONCLUSIONS: Implementation of the hexavalent vaccine in Peru's National Immunization Program has a positive public health cost consequence.

A Dual Strategy-In Vitro and In Silico-To Evaluate Human Antitetanus mAbs Addressing Their Potential Protective Action on TeNT Endocytosis in Primary Rat Neuronal Cells.

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.

[Gaston Ramon and concise history of anatoxins (to the centenary of discovery)].

The article analyzes past history, main milestones of making, research and improvement of anatoxins from works of Gaston Ramon who first proposed 100 years ago method of obtaining reliable vaccine preparation (anatoxin) on the basis of formalin neutralized diphtheria (and then tetanus) toxin (1922-1923) to nowadays scientific achievements in prevention and treatment of these infectious diseases.

Immunomodulation through vaccination as a promising therapeutic strategy to mitigate malaria-related neurocognitive sequelae.

Malaria, an ancient infectious parasitic disease, is caused by protozoa of the genus Plasmodium, whose erythrocytic cycle is accompanied by fever, headache, sweating and chills and a systemic inflammation that can progress to severe forms of disease, including cerebral malaria. Approximately 25% of survivors of this syndrome develop sequelae that may include neurological, neurocognitive, behavioral alterations and poor school performance. Furthermore, some outcomes have also been recorded following episodes of non-severe malaria, which correspond to the most common clinical form of the disease worldwide. There is a body of evidence that neuroinflammation, due to systemic inflammation, plays an important role in the neuropathogenesis of malaria culminating in these cognitive dysfunctions. Preclinical studies suggest that vaccination with type 2 immune response elicitors, such as the tetanus-diphtheria (Td) vaccine, may exert a beneficial immunomodulatory effect by alleviating neuroinflammation. In this viewpoint article, vaccination is proposed as a therapy approach to revert or mitigate neurocognitive deficits associated with malaria.

Low seroprotection against diphtheria and tetanus in Lao adolescents.

OBJECTIVES: There is currently no booster diphtheria or tetanus vaccine for Lao children before adolescence, despite international recommendations. We investigated seroprotection against diphtheria and tetanus among Lao adolescents. METHODS: Seven hundred seventy-nine serum samples were tested for anti-diphtheria and anti-tetanus antibodies. RESULTS: Overall, 25.8% of the adolescents had antibody titers corresponding to protection against diphtheria and 30.9% to sufficient immunity against tetanus. Female participants >16 years were more likely to be protected against diphtheria (p < 0.001) and tetanus (p < 0.029). CONCLUSION: Low protection against diphtheria and tetanus, possibly due to low vaccination coverage or antibody waning, suggests booster doses are warranted before adolescence.

Improved serologic responses to DTaP over Tdap vaccination in adult hematopoietic cell transplant recipients.

BACKGROUND: Hematopoietic cell transplantation (HCT) recipients have reduced antibody titers to tetanus, diphtheria, and pertussis. Tdap is approved for revaccinating adult HCT recipients in the United States, whereas DTaP is not approved in this population. To our knowledge, no studies to date have compared responses to DTaP versus Tdap in adult HCT patients. We conducted a retrospective study comparing responses to DTaP versus Tdap vaccines in otherwise similar adult HCT patients in order to determine if one of these vaccines elicits superior antibody responses. METHODS: We evaluated 43 allogeneic and autologous transplant recipients as a combined cohort and as separate subsets for vaccine specific antibody titers and proportion of strong vaccine responders. Subset analysis focused on the autologous transplant recipients. RESULTS: Higher median antibody titers were found to all vaccine components among DTaP recipients (diphtheria p = .021, pertussis p = .020, tetanus p = .007). DTaP recipients also had more strong responders to diphtheria and pertussis (diphtheria p = .002, pertussis p = .006). Among the autologous HCT recipient subset, there were more strong responders to diphtheria (p = .036). CONCLUSIONS: Our data shows that post-HCT vaccination with DTaP leads to higher antibody titers and more strong responders, which suggests that DTaP is more effective than Tdap in HCT recipients.

Health and Economic Evaluation of Vaccination Against Pertussis in China: A 40-Year Analysis.

OBJECTIVES: This study aimed to evaluate the health and economic impact of diphtheria, tetanus, whole-cell pertussis vaccine (DTwP) and diphtheria-tetanus-acellular pertussis vaccine (DTaP) vaccination on pertussis prevention and control in China during the 40 years from 1978 to 2017. METHODS: We conducted cost-benefit analyses with a decision tree model populated with historical vaccination coverage levels and pertussis incidence and mortality data from before 1978 and during 1978 to 2017. We modeled 40 birth cohorts from birth until death. Costs and benefits were estimated from direct cost and societal perspectives (direct and indirect costs). Costs and benefits were adjusted to 2017 US dollars (USD), and future values were discounted at a 3% annual rate. We calculated net benefit values (net savings) and benefit-cost ratios of pertussis vaccination of children younger than 5 years. We conducted sensitivity analyses by varying key parameters within plausible ranges. RESULTS: Without DTwP and DTaP vaccination, there would be an estimated 115.76 million pertussis cases and 426 650 pertussis deaths in the 40 cohorts. With DTwP/DTaP vaccination, pertussis cases and deaths were decreased by an estimated 92.57% and 97.43%, saving 46 987.81 million USD in direct costs and 82 013.37 million USD from societal perspective. Pertussis vaccination program costs were 2168.76 million USD and 3961.28 million USD from direct cost and societal perspectives. Benefit-cost ratios were 21.67:1 from the direct cost perspective and 20.70:1 from the societal perspective. Sensitivity analyses showed the results to be robust. CONCLUSIONS: Over the lifetime of 40 birth cohorts, China's immunization program is preventing 93% of pertussis cases and 97% of pertussis deaths, resulting in substantial savings to the healthcare system and society.

Tetanus in a vaccinated soil expert - a case report.

BACKGROUND: Tetanus is a life-threatening vaccine-preventable disease found more commonly in tropical climates. It accounted for up to 60 deaths annually until the introduction of the tetanus toxoid. It is now rare in Canada by immunization. This study aims to describe a case of tetanus in Saskatchewan with possible occupational exposure. CASE REPORT: We describe the case of a vaccinated soil expert with tetanus resulting from skin abrasions. This report highlights the early diagnosis, atypical presentation with possible vaccine attenuation and management approach, including acute care, intensive care unit referral, surgical management and physical rehabilitation. We also describe the public health management provided in this case. Despite the severity, the patient returned to his previous home environment with minimal motor sequelae. CONCLUSION: Occupational exposure to tetanus is an important risk, even in regions where the disease is rare. Prevention through vaccination is a key public health intervention that must be encouraged to avoid health complications that are often fatal.

Estimation of vaccination coverage and associated factors in older Mexican adults.

National vaccination programmes recommend the influenza vaccine for older adults, but this population group has the greatest morbidity and mortality from other preventable vaccine diseases. The aim of this article is to estimate the vaccine coverage in adults aged 65 years and older and to analyse the factors that could increase or decrease vaccination uptake probability for the three listed vaccines in the national vaccination programme (influenza, tetanus and diphtheria, and pneumococcus) and the full scheme in Mexico. We conducted an analytical cross-sectional study with 2012, 2018, and 2021 rounds from the National Health and Nutrition Survey, in which we calculated the vaccine coverage estimations and performed multivariable logistic regression models to analyse the factors related to vaccine uptake. Tetanus and diphtheria vaccines had the greatest coverage estimation in all years (59-71%), whereas the pneumococcus vaccine had the lowest (32-53%). Full scheme vaccine coverage decreased from 37.80% to 24.77% in 2012 and 2021, respectively. The National Health Card property, morbidity, being a beneficiary of any health system institution, and use of preventive services increased the probability of vaccine uptake. In conclusion, vaccine coverage in older Mexican adults decreased over time, and the Mexican health system plays a strategic role in immunisation.

Estimating the neural spike train from an unfused tetanic signal of low-threshold motor units using convolutive blind source separation.

BACKGROUND: Individual motor units have been imaged using ultrafast ultrasound based on separating ultrasound images into motor unit twitches (unfused tetanus) evoked by the motoneuronal spike train. Currently, the spike train is estimated from the unfused tetanic signal using a Haar wavelet method (HWM). Although this ultrasound technique has great potential to provide comprehensive access to the neural drive to muscles for a large population of motor units simultaneously, the method has a limited identification rate of the active motor units. The estimation of spikes partly explains the limitation. Since the HWM may be sensitive to noise and unfused tetanic signals often are noisy, we must consider alternative methods with at least similar performance and robust against noise, among other factors. RESULTS: This study aimed to estimate spike trains from simulated and experimental unfused tetani using a convolutive blind source separation (CBSS) algorithm and compare it against HWM. We evaluated the parameters of CBSS using simulations and compared the performance of CBSS against the HWM using simulated and experimental unfused tetanic signals from voluntary contractions of humans and evoked contraction of rats. We found that CBSS had a higher performance than HWM with respect to the simulated firings than HWM (97.5 ± 2.7 vs 96.9 ± 3.3, p < 0.001). In addition, we found that the estimated spike trains from CBSS and HWM highly agreed with the experimental spike trains (98.0% and 96.4%). CONCLUSIONS: This result implies that CBSS can be used to estimate the spike train of an unfused tetanic signal and can be used directly within the current ultrasound-based motor unit identification pipeline. Extending this approach to decomposing ultrasound images into spike trains directly is promising. However, it remains to be investigated in future studies where spatial information is inevitable as a discriminating factor.

Spatial distribution and determinants of tetanus toxoid immunization among pregnant women in Ethiopia using data from Ethiopian demographic and health survey 2016.

INTRODUCTION: Tetanus is a major public health problem caused by clostridium tetani. Although it is vaccine-preventable, the case fatality rate among neonates in areas with poor immunization coverage and limited access to clean deliveries reaches 80-100%. Vaccination of pregnant mothers with the tetanus toxoid (TT) vaccine is the most effective way to protect against neonatal tetanus. This study aimed to examine the spatial distribution and determinants of tetanus toxoid immunization among pregnant mothers using the 2016 EDHS data. METHOD: Secondary analysis of the Ethiopia Demographic and Health Survey 2016 was done to assess the spatial distribution and determinants of tetanus toxoid vaccine among pregnant women in Ethiopia. Spatial autocorrelation analysis and hot spot analysis were used to detect spatial dependency and spatial clustering of the tetanus toxoid vaccine in Ethiopia. Spatial interpolation was used to predict the tetanus toxoid vaccine coverage in unsampled areas. The multilevel binary logistic regression model was fitted to identify factors associated with tetanus toxoid vaccination. An adjusted odds ratio with 95% CI was calculated and used as the measure of association and a p-value less than 0.05 were considered statistically significant. RESULT: From the total of 7043 pregnant women, 42.4% of them have taken at least two doses of tetanus toxoid immunization. Spatial clustering of TT immunization was observed in the Northern, Southwestern and Southwestern parts of Ethiopia. Whereas, low TT coverage was observed in the Eastern and Western parts of the country. Increased ANC visits and the richest economic status favored TT immunization, whereas living in Addis Ababa and Dire Dewa cities decreased the TT immunization coverage. CONCLUSION: The finding of this study reveals that TT immunization had spatial dependency, with the highest immunization coverage observed in the Northern, Southwestern and Southeastern parts of the Country. Thus, geographically targeted interventions should be implemented particularly in the eastern and western parts of the country.

Collaborative study for the calibration of a replacement International Standard for tetanus toxoid for use in flocculation test.

Here we report the results of a study to establish a replacement WHO International Standard (IS) for tetanus toxoid for use in flocculation test. The standard was calibrated in flocculation units (Lf) against the 2nd IS using the Ramon flocculation method. At its 70th meeting in October 2019, WHO ECBS established the material (coded 16/302) as the 3rd WHO IS, with an assigned value of 970 Lf/ampoule from the results of seventeen laboratories across ten different countries. The study also provided an opportunity to assess the use of alternative methods for measuring Lf. Participants were asked to use an in-house Enzyme Linked Immunosorbent Assay (ELISA) developed at NIBSC, or other suitable in-house methods, to determine ELISA-specific Lf values (Lf-eq units are specific only for pre-calibration of antitoxin in the flocculation test) of 16/302 to compare to those of the flocculation test. Nine laboratories participated by performing the NIBSC ELISA, one laboratory performed flocculation by laser light-scattering following an in-house protocol, and three laboratories performed ELISA following in-house protocols. The results intimate that these alternative methods could be useful for monitoring consistency of production at different stages of vaccine manufacturing.