Toxin-specific Monoclonal Antibodies Research Articles

Next Generation Antivenoms and Their Neutralizing Efficacy against Snakebite Envenoming

Snake envenomation is a major neglected tropical disease and remains a serious threat in many countries at the present day. The impoverished rural populations are vulnerable to snakebite envenoming which eventually strengthens the cycle of poverty. The highly diversified components of snake venoms are primarily responsible for severe clinical manifestations in the victim. The only available treatment is the use of animal-derived broad-spectrum polyvalent antivenoms having a very low level of case-specific antibodies. A significant number of drawbacks of this antivenom are possessing a key challenge in snakebite treatments. The recent advancements into the toxin-specific monoclonal antibodies proved to be promising for future envenoming treatments, although its use is still in a naïve stage since it also holds some limitations in neutralizing the complex snake venoms. In the emerging fields of molecular biology along with transcriptomic and proteomic analysis protocols, the snake venom constituents have been well characterized and this holds another promising approach to using DNA immunization for next-generation therapeutics. This strategy is more compatible with the human immune system and exerts the least adverse effects. In this review, the comparative analysis of present polyvalent antivenom and future therapeutic protocols has been discussed.

The Urgent Need to Develop Novel Strategies for the Diagnosis and Treatment of Snakebites.

Snakebite envenoming (SBE) is a priority neglected tropical disease, which kills in excess of 100,000 people per year. Additionally, many millions of survivors also suffer through disabilities and long-term health consequences. The only treatment for SBE, antivenom, has a number of major associated problems, not least, adverse reactions and limited availability. This emphasises the necessity for urgent improvements to the management of this disease. Administration of antivenom is too frequently based on symptomatology, which results in wasting crucial time. The majority of SBE-affected regions rely on broad-spectrum polyvalent antivenoms that have a low content of case-specific efficacious immunoglobulins. Research into small molecular therapeutics such as varespladib/methyl-varespladib (PLA2 inhibitors) and batimastat/marimastat (metalloprotease inhibitors) suggest that such adjunctive treatments could be hugely beneficial to victims. Progress into toxin-specific monoclonal antibodies as well as alternative binding scaffolds such as aptamers hold much promise for future treatment strategies. SBE is not implicit during snakebite, due to venom metering. Thus, the delay between bite and symptom presentation is critical and when symptoms appear it may often already be too late to effectively treat SBE. The development of reliable diagnostical tools could therefore initiate a paradigm shift in the treatment of SBE. While the complete eradication of SBE is an impossibility, mitigation is in the pipeline, with new treatments and diagnostics rapidly emerging. Here we critically review the urgent necessity for the development of diagnostic tools and improved therapeutics to mitigate the deaths and disabilities caused by SBE.

Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62

Tetanus neurotoxin (TeNT) represents a potent toxin that binds to its receptors on neurons and inhibits the release of neurotransmitters. Additionally, its fragments are used to transport pharmacological substances to neuronal cell bodies. The main objective of this study was the development of a suitable model system to study internalization of the TeNT. We have produced a monoclonal antibody (MoAb) specific for TeNT by hybridoma technology, after immunization of BALB/c mice with tetanus toxoid, and have named it T-62. The immunochemical characteristics of MoAb T-62 were tested using ELISA, PAGE and immunoblotting. Finally, we have used an immunohistochemical method to detect specific binding of MoAb T-62 to TeNT bound to PC 12 cells. Our results show that MoAb T-62 is highly specific for TeNT, even when it is bound to its receptor, and that it could be of considerable importance in studies regarding fundamental research on TeNT receptors, intracellular transport of TeNT, as well as retrograde transport of pharmaceutical substances and non-invasive delivery of polypeptides through the blood brain barrier. In addition, MoAb T-62 is an invaluable tool in TeNT vaccine production as it can be used for the detection of reverse toxicity, which could drastically reduce the need to use animals in these experiments.

Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

Molecular and Structural Basis of the Specificity of a Neutralizing Acetylcholine Receptor-mimicking Antibody, Using Combined Mutational and Molecular Modeling Analyses

The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin-specific monoclonal antibody called Malpha2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the Malpha2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp(31) in complementarity determining region 1H is close to, and perhaps interacts with, Arg(33) in the antigen. The use of this pair of proximate residues during the selection procedure yielded three models based on docking calculations. The selected models predicted the proximity of Tyr(49) and/or Tyr(50) in the antibody to Lys(47) in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were submitted to energy minimization in a CHARMM22 force field, and were characterized by a root mean square deviation of 7.0 +/- 2.9 A. Both models display most features of antibody-antigen structures. Since Malpha2-3 also partially mimics some binding properties of nAChR, these structural features not only explain its fine specificity of recognition, but may also further clarify how toxins bind to nAChR.

Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins.

Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.

Inhibition of Staphylococcal Enterotoxin-Driven Lymphocyte Proliferation by Anti-MHC Class II Monoclonal Antibody

Staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules and the V beta region of T cell receptors (TCR) and subsequently induces T cell proliferation. This mitogenicity is the basis of pathological effects seen in food poisoning and toxic shock syndrome. Toxin-specific monoclonal antibodies have previously been shown to be effective in blocking toxin stimulated T cell responses. In this study, a monoclonal antibody, 52BL1, was found to be a potent inhibitor of SEA-, SEB-, SEC1-, SED-, and SEE-induced lymphocyte proliferation assays, which indicates that a single anti-HLA (human leukocyte antigen) class II antibody is effective in blocking the biological effects of these toxins. These results demonstrate the possibility of using anti-HLA class II antibodies in a clinical setting as an antagonist to staphylococcal enterotoxinmediated pathogenesis.

In vitro neutralization of T-2 toxin toxicity by a monoclonal antibody.

A T-2 toxin specific monoclonal antibody, IgG1 K, with a low level of ELISA cross-reactivity to Acetyl T-2, HT-2, and iso T-2 toxins has been produced. The ability of this monoclonal antibody to neutralize the cytotoxicity of T-2 toxin in PHA stimulated cultures of human lymphocytes was determined by the MTT method. The complete neutralization of the toxic effect of 0.02 microM T-2 toxin was obtained with 0.03 microM of MoAb, whereas the 50% neutralizing dose (ND50) was observed at 0.009 microM of MoAb. Partial neutralization was observed with Acetyl T-2 toxin (ND50 = 0.038 microM) and HT-2 (ND50 = 0.94 microM). These results could represent a rational for clinical use of T-2 toxin specific monoclonal antibody in prophylaxis and therapy of T-2 toxemia.

Recombinant Colorimetric Antibodies: Construction and Characterization of a Bifunctional F(ab)2/Alkaline Phosphatase Conjugate Produced in Escherichia coli

We have designed a vector which allows the synthesis in Escherichia coli of bifunctional F(ab)2-alkaline phosphatase conjugates. The vector contains a di-cistronic operon encoding truncated heavy chain (Fd or VH-CH1) of an IgG inserted between residues +6 and +7 of bacterial alkaline phosphatase (PhoA), and the light chain of the same IgG. We demonstrate the utility of this approach with the heavy and light chain domains of a snake toxin-specific monoclonal antibody, M alpha 2-3. We show that the VH-CH1-PhoA hybrid and VL-CL are concomitantly expressed and exported to the periplasm of E. coli where they form a disulfide-linked chimeric protein. The hybrid has the same affinity as M alpha 2-3 for the snake toxin antigen and possesses PhoA enzymatic activity.

Modern approaches to generate antisera against toxic proteins. Use of free synthetic peptides, toxin-specific monoclonal antibodies and genetically engineered toxins

Modern approaches to generate antisera against toxic proteins. Use of free synthetic peptides, toxin-specific monoclonal antibodies and genetically engineered toxins

Production of yeast killer toxin in experimentally infected animals

The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.

Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.

Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein.

We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.

Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.

Shiga-like toxin (SLT-II) was purified to apparent homogeneity from Escherichia coli K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 +/- 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the A1 subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pI of the purified toxin was 5.2. Approximately 1 pg of pure SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and A1 subunits of SLT-II and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification.

Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.

Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.

Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related

Two Cl. difficile toxins were isolated from cultures of Cl. difficile strain VPI 10463. A purification procedure to prepare homogenous Cl. difficile toxins is given. This procedure allows purification of high molecular weight toxins A and B without using immunaffinity chromatography. The main step of the purification is the separation of a partially purified toxin preparation over a FPLC-Mono Q column by anion exchange chromatography. The experimental conditions for a rechromatography were determined to prepare the two major toxic activities as homogenous high molecular weight proteins. Our toxin A has a molecular weight (M r) of ca. 300 kDa and an IP of 4.7. The M r of our toxin B is ca. 250 kDa, the isoelectric focusing gives rise to two bands one at 4.7 and the other at 4.8. The two bands represent charge isomers as have been described for other bacterial toxins. Both toxins differ in cytotoxicity testing by a factor of 1000 but have the same activity when tested in vivo. Toxin specific monoclonal antibodies (mabs) were elicited by separate immunization of mice either with toxin A or toxin B, respectively. All of our mabs cross react with pure toxin A and toxin B when tested by ELISA or Western Blotting. Some mabs strongly cross react indicating that both toxins have major epitopes in common. A hypothesis for the structural and possible functional relatedness between the two toxins is discussed.