Both partial inactivation as well as activation of NF-κB signaling lead to hypertension and chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Activation of NF-κB-signalling is key in the pathogenesis of chronic kidney diseases (CKD). However, a certain level of NF-κB activity is necessary to enable tissue repair. METHODS: To investigate the relationship between activated and inactivated NF-κB signaling on the pathogenesis of CKD using mouse models of NF-κB partial inactivation (mutating cysteine at position 59 of the sixth exon on the NF-κB gene into alanine) and activation (mutating cysteine at position 59 of the sixth exon on the NF-κB gene into serine). RESULTS: The density of CD3, CD8, CD68 positive cells, as well as the expression of IL-6, TRAF-1, and NAF-1 in the kidney tissues of NF-κBC59A mice were reduced, whereas an opposing pattern was observed in the NF-κBC59S mice. Blood pressure, kidney fibrosis (analyzed by PAS-, Masson trichrome-, and Sirius-Red-staining as well as α-SMA immunofluorescence), serum creatinine and urinary albumin-to-creatinine-ratio are markedly increased in NF-κB activated and inactivated mice compared to controls. Transmission electron microscopy indicated that the glomerular basement membrane was thicker in both NF-κBC59A and NF-κBC59S mice compared to wild-type mice. CONCLUSIONS: Using mice models with partially activated and inactivated NF-κB pathways suggests that there is an apparently U-shaped relationship between blood pressure, kidney function as well as morphology and the activation of the NF-κB pathway. A certain optimal activity of the NF-κB pathway seems to be important to maintain optimal kidney function and morphology.

Multiple-breath washout to detect lung disease in patients with inborn errors of immunity.

Background: Pulmonary manifestations are the major cause of morbidity and mortality in patients with inborn errors of immunity (IEI). New and more sensitive diagnostic methods can potentially lead to earlier recognition and treatment of IEI lung disease and improve outcome. The aim of this study was to compare multiple-breath washout (MBW) and spirometry in patients with IEI and cystic fibrosis (CF) as well as healthy controls (HC) and to evaluate the sensitivity of lung clearance index (LCI) to assess lung disease in IEI. Methods: IEI patients (n=114) were recruited from our paediatric and adult immunodeficiency outpatient clinics and compared to age-matched CF patients (n=114) and HC (n=114). MBW measurements and spirometry were performed in the study participants, and MBW testing was repeated after 63-707 days in IEI patients (n=70). Results: The LCI was significantly higher in IEI patients than in HC (p<0.001) and significantly lower than in CF patients (p<0.001). The forced expiratory volume in 1 s (FEV1) z-score was significantly lower in IEI patients than in HC (p<0.01) and significantly higher than in CF patients (p<0.01). LCI and FEV1 z-score correlated moderately negatively in the total cohort, the IEI group and the CF group. Nineteen (20.7%) of 92 IEI patients and 35 (33.3%) of 105 CF patients had an elevated LCI but a normal FEV1 z-score. After a median of 364 days, the median LCI of 70 IEI patients increased significantly by 0.2. Conclusion: MBW is useful to detect lung disease in IEI and is more sensitive than spirometry.

Only bioactive forms of PTH (n-oxPTH and Met18(ox)-PTH) inhibit synthesis of sclerostin - evidence from in vitro and human studies.

Sclerostin (SOST) is produced by osteocytes and is known as a negative regulator of bone homeostasis. Parathyroid hormone (PTH) regulates calcium, phosphate as well as vitamin D metabolism, and is a strong inhibitor of SOST synthesis in vitro and in vivo. PTH has two methionine amino acids (positions 8 and 18) which can be oxidized. PTH oxidized at Met18 (Met18(ox)-PTH) continues to be bioactive, whereas PTH oxidized at Met8 (Met8(ox)-PTH) or PTH oxidized at Met8 and Met18 (Met8, Met18(di-ox)-PTH) has minor bioactivity. How non-oxidized PTH (n-oxPTH) and oxidized forms of PTH act on sclerostin synthesis is unknown. The effects of n-oxPTH and oxidized forms of PTH on SOST gene expression were evaluated in UMR106 osteoblast-like cells. Moreover, we analyzed the relationship of SOST with n-oxPTH and all forms of oxPTH in 516 stable kidney transplant recipients using an assay system that can distinguish in clinical samples between n-oxPTH and the sum of all oxidized PTH forms (Met8(ox)-PTH, Met18(ox)-PTH, and Met8, Met18(di-ox)-PTH). We found that both n-oxPTH and Met18(ox)-PTH at doses of 1, 3, 20, and 30 nmol/L significantly inhibit SOST gene expression in vitro, whereas Met8(ox)-PTH and Met8, Met18(di-ox)-PTH only have a weak inhibitory effect on SOST gene expression. In the clinical cohort, multivariate linear regression showed that only n-oxPTH, but not intact PTH (iPTH) nor oxPTH, is independently associated with circulating SOST after adjusting for known confounding factors. In conclusion, only bioactive PTH forms such as n-oxPTH and Met18(ox)-PTH, inhibit SOST synthesis.

The Degree of Prepregnancy Vitamin D Deficiency Is Not Associated With Gestational Diabetes in Women Undergoing ART.

Context: Gestational diabetes mellitus (GDM) is a common pregnancy complication, particularly in women undergoing assisted reproductive technology (ART). An association of GDM with vitamin D in women conceiving naturally has been described; however, studies have yielded heterogeneous results. Objective: To analyze the association between prepregnancy total and free vitamin D and GDM incidence in women undergoing ART. Methods: Post hoc analysis of a prospective study at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha, China. Total and free vitamin D were measured 1 day before embryo transfer. The patients were screened for GDM using the oral glucose tolerance test. Results: A total of 1593 women were included in the study, among whom 256 (16.1%) developed GDM. According to international guidelines for total 25-hydroxyvitamin D [25(OH)D], 47 (2.9%) patients had sufficient (≥30 ng/mL) levels, while 696 (43.7%) were insufficient (20 to <30 ng/mL) and 850 (54.4%) were deficient (<20 ng/mL). Comparing GDM and non-GDM patients, there was no significant difference in total nor free vitamin D levels (P = .340 and .849). Similarly, analysis of GDM rates by quintiles of total and free 25(OH)D showed no significant association in one-way ANOVA (P = .831 and .799). Multivariate logistic regression, considering age, BMI, and fasting glucose, also did not show a significant influence of the 2 vitamin D forms on GDM incidence (P = .266 and .123 respectively). Conclusion: In this relatively vitamin D deficient/insufficient ART cohort, the degree of neither total nor free vitamin D deficiency before pregnancy was associated with the occurrence of GDM.

Immune response of heterologous versus homologous prime-boost regimens with adenoviral vectored and mRNA COVID-19 vaccines in immunocompromised patients.

Due to rare but major adverse reactions to the AstraZeneca adenoviral ChAdOx1-S-nCoV-19 vaccine (ChAd), German health authorities recommended adults under 60 who received one dose of ChAd, to receive a second dose of the BioNTech mRNA BNT162b2 vaccine (BNT) as a booster. Studies in the general population suggest an enhanced efficacy of the heterologous (ChAd-BNT) compared to the homologous (BNT-BNT) vaccination regimen. However, an analysis of the efficacy in patient populations with a high risk of severe COVID-19 due to acquired immunodeficiency is still missing. We therefore compared both vaccination regimens in healthy controls, patients with gynecological tumors after chemotherapy, patients on dialysis and patients with rheumatic diseases concerning the humoral and cellular immune response. The humoral and cellular immune response differed substantially in healthy controls compared to patients with acquired immunodeficiency. Overall, the most significant differences between the two immunization regimens were found in neutralizing antibodies. These were always higher after a heterologous immunization. Healthy controls responded well to both vaccination regimens. However, the formation of neutralizing antibodies was more pronounced after a heterologous immunization. Dialysis patients, on the other hand, only developed an adequate humoral and particularly cellular immune response after a heterologous immunization. Tumor and rheumatic patients also - to a weaker extent compared to dialysis patients - benefited from a heterologous immunization. In conclusion, the heterologous COVID-19 vaccination regimens (ChAd-BNT) seem to have an advantage over the homologous vaccination regimens, especially in immunocompromised patients such as patients with end-stage kidney disease treated with hemodialysis.

Parental sex-dependent effects of either maternal or paternal eNOS deficiency on the offspring's phenotype without transmission of the parental eNOS deficiency to the offspring.

Background: Preclinical animal studies and clinical studies indicate that both maternal as well as paternal genetic alterations/gene defects might affect the phenotype of the next-generation without transmissions of the affected gene. Currently, the question of whether the same genetic defect present in the mother or father leads to a similar phenotype in the offspring remains insufficiently elucidated. Methods: In this head-to-head study, we crossbred female and male mice with heterozygous endothelial eNOS knockout (eNOS+/-) with male and female wild-type (wt) mice, respectively. Subsequently, we compared the phenotype of the resulting wt offspring with that of wt offspring born to parents with no eNOS deficiency. Results: Wt female offspring of mothers with heterozygous eNOS showed elevated liver fat accumulation, while wt male offspring of fathers with heterozygous eNOS exhibited increased fasting insulin, heightened insulin levels after a glucose load, and elevated liver glycogen content. By quantitative mass-spectrometry it was shown that concentrations of six serum metabolites (lysoPhosphatidylcholine acyl C20:3, phosphatidylcholine diacyl C36:2, phosphatidylcholine diacyl C38:1, phosphatidylcholine acyl-alkyl C34:1, phosphatidylcholine acyl-alkyl C36:3, and phosphatidylcholine acyl-alkyl C42:5 (PC ae C42:5) as well as four liver carbon metabolites (fructose 6-phosphate, fructose 1,6-bisphosphate, glucose 6-phosphate and fumarate) were different between wt offspring with eNOS+/- mothers and wt offspring with eNOS+/- fathers. Importantly, fumarate was inversely correlated with the liver fat accumulation in female offspring with eNOS+/- mothers and increased liver glycogen in offspring of both sexes with eNOS+/- fathers. The qRT-PCR results revealed that the gene expression patterns were different between wt offspring with eNOS+/- mothers and those offspring with eNOS+/- fathers. Different gene expression patterns were correlated with different observed phenotypic changes in male/female offspring born to mothers or fathers with a heterozygous eNOS genotype. Conclusion: The identical parental genetic alteration (heterozygous eNOS deficiency), without being passed on to the offspring, results in distinct metabolic, liver phenotype, and gene expression pattern variations depending on whether the genetic alteration originated from the father or the mother.

Effectiveness and analysis of factors predictive of discharge to home in a 4-year cohort in a residential transitional care unit.

OBJECTIVE: The aim of this study was to evaluate the effectiveness and identify factors predictive of home discharge in a cohort of patients admitted to the residential Transitional Aged Care Program (r-TACP) after a stay in an acute hospital. METHODS: A retrospective observational cohort study of patients admitted to a single r-TACP unit between 1 January 2014 and 31 December 2017 was carried out. Baseline patient characteristics and discharge outcomes were analyzed. RESULTS: Three hundred sixty-nine patients were admitted during the study period. The discharge outcomes were as follows: 68% returned home, 17% went onto residential care, 14% were readmitted to hospital, and 1% died. Factors associated with not returning home were increased age, increased comorbidities, and lower Barthel Index on admission to the r-TACP. CONCLUSION: Our r-TACP is an effective program that successfully returns the majority (67.8%) of older patients home after an acute hospital admission. Older patients with greater comorbidities and poorer baseline functional status in our program were less likely to return home.

Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.

Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E.

BACKGROUND: Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. RESULTS: In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. CONCLUSION: These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library.

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.

A functional dual-coated (FDC) microtiter plate method to replace the botulinum toxin LD50 test.

Conventional capture ("Sandwich") ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtiter plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin's Hc domain, it was possible to develop a highly sensitive (130 aM LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g., 1.2 M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favorable buffer containing zinc and DTT reduced the interchain disulfide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1-206) substrate. A neoepitope antibody specific for the newly exposed Q(197) epitope was used to quantify the cleaved SNAP25(1-197). The assay's requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments, and stressed samples containing partially or fully denatured material. This is the first known immunobiochemical assay that correlates with in vivo potency and provides a realistic alternative.

Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin.

BACKGROUND: Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use. RESULTS: Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8. CONCLUSIONS: The presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate antibody fragments with therapeutic potential.

Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: detection with a new neoepitope endopeptidase immunoassay.

Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.

New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, C1 and E intra-cellularly cleave SNAP25 and/or Syntaxin (type C1 only) resulting in a flaccid paralysis. Although highly sensitive, robust in vitro endopeptidase immunoassays have been developed for some serotypes, an endopeptidase immunoassay for type C1 has not previously been described. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and a new specific antibody to the SNAP25(191-198) octapeptide epitope that becomes exposed following cleavage by type C1 toxin. The highly specific nature of the detecting antibody was illustrated by the failure of anti-SNAP25(191-198) to recognise the type A cleavage product which differs by just one amino acid residue. Conversely, anti-SNAP25(190-197), which recognises the type A cleavage product, fails to cross react with the type C1 toxin cleavage product. Utilising Syntaxin(232-266) peptide substrate, and a specific antibody to the cleavage product epitope, Syntaxin(254-261), it was also possible to develop an endopeptidase immunoassay. Assay sensitivities allowed the detection of less than 0.1 LD(50)/ml (25 pg/ml) of type C1 haemagglutinin-complexed toxin. The assay failed to detect toxin serotypes A, B, D, E, F or G and therefore also provides an alternative highly specific in vitro identity test. In the absence of trypsin inhibitors, the assay is also capable of detecting 2 pg/ml of trypsin activity, or trypsin like contaminants. These new immunoassays will therefore provide highly specific tools for monitoring botulinum toxin light chain endopeptidase activity and serotype identity.

An improved method for development of toxoid vaccines and antitoxins.

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylation with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600-times higher than those produced with formaldehyde toxoid, despite generating equivalent ELISA antitoxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxins structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins and immunogens.