Development Of Antitoxin Research Articles

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.

The Relative Mechanism of Tetrodotoxin: From Poison to Drug

Tetrodotoxin (TTX), an exceptionally potent neurotoxin initially found in pufferfish ovaries in 1909, is renowned for its extreme toxicity to humans, surpassing cyanide. This research delves into TTX, from its action on sodium channels to its potential as a therapeutic agent. TTX primarily blocks voltage-gated sodium channels, halting nerve cell electrical signals, but its binding varies among channel subtypes. TTX's role in pain, especially cancer-related pain, is intriguing, but its medical use is in the early stages and needs more research. TTX poisoning treatment is limited to emesis, activated charcoal, sodium bicarbonate, fluid replacement, and respiratory support. This research will conclude by highlighting TTX's potential in future research, such as antitoxin development and medical applications. TTX, once feared, may become a valuable medical tool, offering hope and healing. A deep understanding of the mechanism of action of TTX is beneficial for the development of corresponding applications in different medical fields.

Neuronal Cellular Nanosponges for Effective Detoxification of Neurotoxins.

Neurotoxins attack and destruct the nervous system, which can cause serious health problems and security threats. Existing detoxification approaches, such as antibodies and small molecule antidotes, rely on neurotoxin's molecular structure as design cues and require toxin-specific development for each type of toxins. However, the enormous diversity of neurotoxins makes such structure-based development of antitoxin particularly challenging and inefficient. Here, we report on the development and use of neuronal membrane-coated nanosponges (denoted "Neuron-NS") as an effective approach to detoxifying neurotoxins. Specifically, Neuron-NS act as neuron decoys to lure neurotoxins, bind with and neutralize the toxins, and thus block them from attacking the host neuron cells. These nanosponges detoxify neurotoxins regardless of their molecular structures and therefore can overcome the challenge posed by toxin structural diversity. In the study, we fabricate Neuron-NS by coating the membrane of Neuro-2a cells onto polymeric cores. Meanwhile, we select tetrodotoxin (TTX) as a model neurotoxin and demonstrate the detoxification efficacy of the Neuron-NS in a cytotoxicity assay, a calcium flux assay, and a cell osmotic swelling assay in vitro. Additionally, in mouse models of TTX intoxication, the Neuron-NS significantly enhance mouse survival in therapeutic and prophylactic regimens without showing acute toxicity. Overall, the Neuron-NS contribute to the current detoxification arsenal with the potential to treat various injuries and diseases caused by neurotoxins.

Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats.

Botulinum neurotoxins (BoNT) are potential biothreat agents due to their high lethality, potency, and ease of distribution, thus the development of antitoxins is a high priority to the US government. This study examined pre-clinical pharmacokinetic studies in rats of four oligoclonal anti-BoNT mAb-based therapeutics (NTM-1631, NTM-1632, NTM-1633, NTM-1634) for five BoNT serotypes (A, B, E, C, and D). NTM-1631, NTM-1632, and NTM-1633 each consist of three IgG1 mAbs, each with a distinct human or humanized variable region which bind to distinct epitopes on BoNT serotype A, B, or E respectively. NTM-1634 consists of four human immunoglobulin G1 (IgG1) mAbs binding BoNT C/D mosaic toxins. The mechanism of these antitoxins requires that three antibodies simultaneously bind toxin to achieve rapid clearance. Rats (total 378) displayed no adverse clinical signs attributed to antibody treatment from any of the antitoxins. Pharmacokinetic evaluation demonstrated that the individual mAbs are slowly eliminated, exhibiting dose-dependent exposure and long elimination half-lives ranging from 6.5 days to 10 days. There were no consistent differences observed between males and females or among the individual antibodies in each formulation in half-life. Anti-drug antibodies (ADA) were observed, as expected for human antibodies administered to rats. The results presented were used to support the clinical investigation of antibody-based botulism antitoxins.

From Fangs to Pharmacology: The Future of Snakebite Envenoming Therapy.

The snake is the symbol of medicine due to its association with Asclepius, the Greek God of medicine, and so with good reasons. More than 725 species of venomous snakes have toxins specifically evolved to exert potent bioactivity in prey or victims, and snakebites constitute a public health hazard of high impact in Asia, Africa, Latin America, and parts of Oceania. Parenteral administration of antivenoms is the mainstay in snakebite envenoming therapy. However, despite well-demonstrated efficacy and safety of many antivenoms worldwide, they are still being produced by traditional animal immunization procedures, and therefore present a number of drawbacks. Technological advances within biopharmaceutical development and medicinal chemistry could pave the way for rational drug design approaches against snake toxins. This could minimize the use of animals and bring forward more effective therapies for snakebite envenomings. In this review, current stateof- the-art in biopharmaceutical antitoxin development is presented together with an overview of available bioinformatics and structural data on snake venom toxins. This growing body of scientific and technological tools could define the basis for introducing a rational drug design approach into the field of snakebite envenoming therapy.

Unique Substrate Recognition Mechanism of the Botulinum Neurotoxin D Light Chain

Botulinum neurotoxins are the most potent protein toxins in nature. Despite the potential to block neurotransmitter release at the neuromuscular junction and cause human botulism, they are widely used in protein therapies. Among the seven botulinum neurotoxin serotypes, mechanisms of substrate recognition and specificity are known to a certain extent in the A, B, E, and F light chains, but not in the D light chain (LC/D). In this study, we addressed the unique substrate recognition mechanism of LC/D and showed that this serotype underwent hydrophobic interactions with VAMP-2 at its V1 motif. The LC/D B3, B4, and B5 binding sites specifically recognize the hydrophobic residues in the V1 motif of VAMP-2. Interestingly, we identified a novel dual recognition mechanism employed by LC/D in recognition of VAMP-2 sites at both the active site and distal binding sites, in which one site of VAMP-2 was recognized by two independent, but functionally similar LC/D sites that were complementary to each other. The dual recognition strategy increases the tolerance of LC/D to mutations and renders it a good candidate for engineering to improve its therapeutic properties. In conclusion, in this study, we identified a unique multistep substrate recognition mechanism by LC/D and provide insights for LC/D engineering and antitoxin development.

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

On Viruses, Discovery, and Recognition

This year's Nobel Prize in Physiology or Medicine rewards the discoverers of two viruses that cause major afflictions of humankind. Identifying human papilloma virus (HPV) and human immunodeficiency virus (HIV) might now appear to have been simple, but the way forward was far from obvious at the time.

Influence of Carrier Proteins on the Immunologic Response to Haptenic Antitetrodotoxin Vaccine

Tetrodotoxin (TTX) is a haptenic, highly toxic neurotoxin with no specific antidote available yet. Anti-TTX vaccine is being studied for antitoxin development. The effectiveness of the carrier protein in eliciting TTX-specific antibody response was comparatively studied. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), and bovine serum albumin (BSA) chemically to form artificial antigens TTH-TTX, LPH-TTX, TT-TTX, DT-TTX, and BSA-TTX, respectively, with which BALB/c mice were immunized, and the antibody response and antitoxic efficacy were detected. The serum anti-TTX antibody response and antitoxic efficacy varied markedly with adopted carrier protein. TTH-TTX elicited the best and BSA-TTX the worst TTX-specific antibody response. The proportion of the immunized mice surviving a 3x lethal dose (LD) dose of TTX challenge was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX conjugates, respectively. The rank order of total efficacy of carrier protein for both anti-TTX antibody response and antitoxic effect was TTH > TT > LPH > DT > BSA. As a result of formaldehyde treatment in coupling of TTX carriers, the relative immunogenicity of TTX vs carrier, that is, the ratio of TTX- to carrier-specific antibody response, evidently varied with respective carrier adopted, in a rank order of TT > BSA > TTH > DT > LPH. The results suggest that the carrier protein used in haptenic TTX vaccine is greatly important in eliciting potent anti-TTX antibody, and both TTH and TT are the preferred carriers for development of excellent experimental TTX vaccine.

Acute plant poisoning and antitoxin antibodies.

Plant poisoning is normally a problem of young children who unintentionally ingest small quantities of toxic plants with little resulting morbidity and few deaths. In some regions of the world, however, plant poisonings are important clinical problems causing much morbidity and mortality. While deaths do occur after unintentional poisoning with plants such as Atractylis gummifera (bird-lime or blue thistle) and Blighia sapida (ackee tree), the majority of deaths globally occur following intentional self-poisoning with plants such as Thevetia peruviana (yellow oleander) and Cerbera manghas (pink-eyed cerbera or sea mango). Antitoxins developed against colchicine and cardiac glycosides would be useful for plant poisonings--anti-digoxin Fab fragments have been shown to be highly effective in T. peruviana poisoning. Unfortunately, their great cost limits their use in the developing world where they would make a major difference in patient management. Therapy for some other plant poisonings might also benefit from the development of antitoxins. However, until issues of cost and supply are worked out, plant antitoxins are going to remain a dream in many of the areas where they are now urgently required.

Anthrax toxin receptor proteins

Anthrax toxin is a key virulence factor for Bacillus anthracis, the causative agent of anthrax. Here we discuss what is known about the anthrax toxin receptor (ATR), the cellular receptor for anthrax toxin, and how this information is being used to develop treatments for anthrax as well as to understand aspects of cancer. ATR was identified recently as a type I transmembrane protein with unknown function that contains an extracellular integrin-like inserted (I) domain. The ATR I domain contains the toxin binding site, and a soluble form of this domain was shown to serve as an effective antitoxin to protect cultured cells from toxin action. ATR is encoded by the tumor endothelial marker 8 ( TEM8) gene, which is selectively up-regulated during blood vessel formation and in tumor vasculature, raising the possibility that this protein normally functions in angiogenesis. Therefore, identification of the cellular receptor for anthrax toxin has made possible new avenues of research in the areas of anthrax pathogenesis, antitoxin development, and cancer biology.

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration.

Development of antitoxin with each of two complementary fragments of Clostridium botulinum type B derivative toxin

Two fragments with molecular weights of 111,000 (fragment I) and 59,000 (fragment II) were separated from each other by gel filtration of dithiothreitol and urea-treated, trypsinized derivative toxin (molecular weight, 170,000) of the proteolytic Okra strain of Clostridium botulinum type B on a column of Sephadex G-200 (superfine) with a buffer containing dithiothreitol and urea. Upon removal of dithiothreitol and urea by dialysis, the two fragments reassembled to reconstruct the derivative toxin molecule. Both fragments were immunogenic, and both anti-fragments neutralized type B toxin. The neutralizing activities of both anti-fragment I and anti-fragment II were, however, lower than that of the anti-derivative toxin, suggesting that the molecular integrity of derivative toxin is essential for sufficient production of the neutralizing antibody. The immunological difference found between type B toxin from a proteolytic strain and that from a nonproteolytic strain was ascribed to the antigenic difference of fragment I.

Quantitative Studies in Diphtheria Prophylaxis: An Attempt to Derive a Mathematical Characterization of the Antigenicity of Diphtheria Prophylactic

Examined quantitatively, antibody responses of animals and children to inoculatioiis of different forms of diphtheria prophylactic vary greatly. The dose-response curves from such materials, however, do show a similar pattern, and there is a great variation between different forms of prophylactic in dosage required to induce some arbitrary level of response (Jerne & Maal0e, 1949). As Jerne & Wood (1949) point out, an assay of a test preparation (T.P.) in terms of a standard preparation (S.P.) is strictly valid only if the less potent preparation behaves as though it were a dilution of other in a completely inert dilutent. The relative potency of T.P. in terms of S.P., defined as ratio of doses required to produce a given response, is then independent of dose level of response at which it is measured. They continue . . this is only definition of relative potency that would normally be regarded by bio-assayist as satisfactory . . . . An instance of current interest in which this assumption does not hold is assay of diphtheria and tetanus toxoids in commercial products containing aluminium hydroxide, using as S.P. a reference sample of highly purified toxoid . . . doseresponse curves of two preparations have different upper asymptotes and cannot be described by same form .... The assay is thus invalid. Here then we have problem; two preparations have a property in common, viz. ability to cause development of antitoxin when injected, but one cannot be expressed quantitatively in terms of other in usual way. The present communication is concerned with (a) an attempt to overcome this difficulty by finding antigenicity equations applicable to all types of diphtheria prophylactic (and probably other antigeiis)

Antibody formation in early infancy against diphtheria and tetanus toxoids

Summary A study was made of the development of antitoxin after two injections of combined diphtheria and tetanus toxoids in a group of young infants. It was found that the production of tetanus antitoxin is as good in the early months of life as later, and this finding denotes that the mechanism for the production of antibodies is well developed in young infants. With diphtheria antigen, the development of antitoxin is definitely defective in a considerable number during the first 6 months of life. This impairment of antibody formation is related to the presence of passive immunity in the resistant infants and appears specific for diphtheria. The application of these findings to the practical immunization of infants to diphtheria, tetanus, and whooping cough was discussed.

An Electrophoretic Study of Tetanus Antitoxin Sera

In a previous paper we described an electrophoretic analysis of several antipneumococcal horse sera that showed antibody activity in the γ-component. Since then 2 of the horses have been used to produce tetanal antitoxin. Subsequent electrophoretic analyses detected the appearance of a new component apparently associated with the freshly developed antitoxic activity. Early in September, 1939, horses No. 9514 and No. 6225 began to receive tetanal toxin instead of Types 1 and 2 pneumococcal antigens. Three months later they had high antitoxin-titers. Electrophoretic analyses of sera taken then, and before immunization with toxin had begun, are reproduced in Figs. 1 to 4. Only traces of antipneumococcal precipitin could be found in the sera taken in December and areas underlying the γ-peaks had obviously decreased greatly. With the development of antitoxin there had appeared, however, a strong new component (labeled T) with a mobility, -2.2 × 10-5 cm sec-1 volts-1, midway between β and γ. The same component was present in the strongly antitoxic serum of horse No. 756, which had never been used before for antibody-production, and in the sera of other horses producing tetanal antitoxin.

Active immunization against tetanus infection with refined tetanus toxoid: Bergey, D. H., and Etris, S.: J. Immunol. 31: 363, 1936

In earlier papers (1, 2, 3, 4) the authors have reported on the immunity developed by unprocessed tetanus toxoid in guinea pigs following the subcutaneous injection of the toxoid. When the unprocessed tetanus toxoid was tested on human beings by intracutaneous and subcutaneous injections it was found to produce annoying local reactions. This experience led to the development of the refined alum precipitated toxoid. The refined tetanus toxoid was prepared by precipitating the unprocessed toxoid with aluminum-potassium sulphate and washing the precipitate several times with sterile normal saline solution. The purified toxoid was then brought to its original volume with sterile saline solution following the method of precipitation and refinement of diphtheria toxoid devised by Wells, Graham and Havens (5). Antitoxin Development in Guinea Pigs. A study of the comparative antigenic value of the unprocessed toxoid and the refined toxoid was carried out in guinea pigs weighing 350 grams.

Active Immunization Against Tetanus Infection with Refined Tetanus Toxoid

Abstract In earlier papers (1, 2, 3, 4) the authors have reported on the immunity developed by unprocessed tetanus toxoid in guinea pigs following the subcutaneous injection of the toxoid. When the unprocessed tetanus toxoid was tested on human beings by intracutaneous and subcutaneous injections it was found to produce annoying local reactions. This experience led to the development of the refined alum precipitated toxoid. The refined tetanus toxoid was prepared by precipitating the unprocessed toxoid with aluminum-potassium sulphate and washing the precipitate several times with sterile normal saline solution. The purified toxoid was then brought to its original volume with sterile saline solution following the method of precipitation and refinement of diphtheria toxoid devised by Wells, Graham and Havens (5). Antitoxin Development in Guinea Pigs. A study of the comparative antigenic value of the unprocessed toxoid and the refined toxoid was carried out in guinea pigs weighing 350 grams.

VII. THE COURSE OF THE DEVELOPMENT OF IMMUNITY IN SCARLET FEVER

In a study 1 of the changes in skin sensitivity to scarlatinal streptococcus filtrate toxin, and the development of neutralizing antitoxin following injections of the filtrate, it was found that the decrease in skin sensitivity appeared relatively quickly, and that antitoxin was not usually demonstrable until a week or two later. The rapid change in skin sensitivity resembled desensitization of an anaphylactically hypersensitive tissue following injection of antigen, and appeared not to be dependent on the later formation of specific antibodies. In the present paper, observations on the changes in skin sensitivity and the development of antitoxin in the course of clinical scarlet fever are reported. The reaction to the Dick test in scarlet fever, according to a number of reports, is modified by the disease and tends to become negative during convalescence. Before the onset, the reaction has been positive in almost all those tested, and, in many instances,