The future of glycoprotein VI as an antithrombotic target.

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

[Envenomations VI. Scorpionism in Africa]. / Envenimations vi--le scorpionisme en Afrique.

Scorpionism is a serious public health problem in North Africa, sub-Saharan countries, and South Africa. The number of species involved is small, but some have large geographic areas of distribution. Fatal outcomes sometimes occur in children and young adults. In most cases the only symptom after a scorpion sting is intense, persistent pain. Appearance of digestive symptoms within a few hours after the sting (5% of cases) is a sign of severe envenomation. These symptoms can be followed by pulmonary edema and cardiovascular manifestations (arrhythmia and hypotension). Early death can occur due to cardiovascular failure (1 to 2% of untreated cases). Progression is rapid and, after a period of 24 hours, the patient is out of danger and will recover quickly with not sequels. Specific (serotherapy) and symptomatic treatment must be initiated as soon as possible after the sting. Envenomation of a child by a dangerous adult scorpion (more than 5 cm long) in summer should be considered as a medical emergency. Declining production of antivenom is a disturbing development.

Using a recombinant bispecific antibody to block Na+ -channel toxins protects against experimental scorpion envenoming.

In recent years, several molecular engineering methods of designing bispecific antibodies in various formats have been developed. Tandem-scFvs comprising two scFvs fused together via a peptide are 55-kDa molecules, and are one of the most promising and most straightforward approaches to bispecific antibody production. We report an attempt to design more effective antivenoms to the Androctonus australis scorpion using murine scFvs as building blocks to create a unique bispecific molecule that neutralizes the potent neurotoxins AahI and AahII. The tandem-scFv was produced in recombinant bacteria, purified by immobilized metal ion affinity chromatography, and analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, mass spectrometry, and direct and competitive radioimmunoassay. In vivo, it neutralized the binding of the AahI and AahII toxins to their receptor, and protected mice against experimental envenomation. The findings reported here highlight the potential of recombinant antibody fragments for protecting against scorpion venom toxicity.

[Recombinant antibodies: a new application in scorpion envenomation?]. / Anticorps recombinants: vers un renouveau de la sérothérapie anti-scorpionique?

Serotherapy is the only specific treatment for envenomation. The antibodies are obtained after the purification of serum from hyperimmunised horses and are used after fragmentation in the form of polyclonal Fab or F(ab)'2. The anti-venom sera are heterogeneous, and their protective effect is often weak. The administration of these preparations induces risks of immediate or delayed side effects: hypersensitivity reactions, anaphylactic shock and serum sickness. This observation led us to develop new forms of antibodies produced by molecular engineering, capable of specifically neutralizing the neurotoxins responsible for the toxicity of the venom of Androctonus australis Hector. The recombinant antibody fragments are more homogeneous than conventional antivenoms and perfectly characterized in terms of specific activity. The method used to obtain them eliminates the risk of transmission by non-conventional transmissible agents. The earliest results confirm the importance of these new molecules (scFv recombinant Fab, diabody, triabody) and their ability to neutralize the action of scorpion neurotoxins. They could open the path to a new generation of more homogeneous antivenoms that are better tolerated and have well-characterized intrinsic properties.

Design and evaluation of a diabody to improve protection against a potent scorpion neurotoxin.

Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner. In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals. The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography. It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M). The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations. Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI. In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations.

[Rapid immunotitration of individual toxins from Androctonus australis venom]. / Immunodosage rapide des toxines individuelles du venin d'Androctonus australis.

Passive immunotherapy against scorpion envenomations is facilitated by the preliminary titration of circulating toxins in envenomed patients. Currently, routinely used ELISA tests allow only the titration of the whole venom, without reference to the toxins which compose the venom and spread variably within the tissue. Taking as a model one of the three toxins responsible for the lethal effects of Androctonus australis hector (Aahl) venom, we developed an ELISA sandwich test based on a fragment of recombining antibody (scFv) consisting of the variable chains of the monoclonal IgG 9C2 coupled to a decapeptide showing high affinity for streptavidine. Conjugate scFvlStrep-tag was prepared by genetic engineering. It was produced in the periplasm of recombining bacteria, in a reproducible way, in a soluble form, at low cost and with an output, after purification, of 0.8 mg/L of bacterial culture. The recombinant protein, of small size (28 kDa), is bifunctional. It preserves a very high affinity for the toxin Aah I (Kd of 2.3 10(-10) M, very close to that of IgG 9C2), yet recognises streptavidine and its conjugate (streptavidine-peroxidase). The titration of the Aahl toxin used an ELISA sandwich test in which the toxin was captured in a specific way by a monoclonal antibody; the immunocomplexes were then detected by recombinant immunoconjugate, thus conferring a high specificity on titration. The test is quick (90 mn), reproducible and sensitive, with a limit of detection of 0.6 toxin (ng.ml-1). This method could be extended to two other lethal toxins of the venom of the scorpion Androctonus australis hector and to those of other species. New perspectives are thus possible for the diagnosis of the envenomations.

Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatography on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3 x 10(-10) M). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate.

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.

A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector.

Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning. A single-chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations.

Production and characterization of a bivalent single chain Fv/alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis.

A102 is a monoclonal antibody raised against the hemocyanin of the Tunisian scorpion Androctonus australis. It is directed against the subunit Aa6 and does not cross-react when tested against a variety of similar scorpion hemocyanins. Here, we report the construction of a plasmid encoding a recombinant enzyme-linked antigen-binding protein with the antigen-binding specificity of antibody A102. The DNA fragments encoding the variable domains of A102 were inserted into a prokaryotic expression vector so as to produce a single chain antibody variable fragment (scFv) fused to the bacterial alkaline phosphatase. The fusion protein preserved the IgG binding and alkaline phosphatase activities. Immunoelectron microscopic analysis showed that the recombinant protein bound antigen bivalently as is the case for natural antibodies. Crude preparations containing the conjugate were used in a rapid visual immunoassay for the specific detection of A. australis hemocyanin, using a droplet of hemolymph removed from live animals by puncture. The simplicity of the test made it suitable for the direct identification of animals belonging to this species. It could be useful in areas where A. australis, the most dangerous African scorpion, is found with other species from which it is not easy to distinguish using morphological criteria.

Production of a functional anti-scorpion hemocyanin scFv in Escherichia coli.

A58 is a murine monoclonal antibody raised against the internal dimeric subunit A alpha 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH--VL structure-function relationships of antibodies.

[PCR: principles and prospects in clinical biology]. / PCR: principe et perspectives en biologie clinique.

In the short time it has been available, the Polymerase Chain Reaction (PCR) has been rapidly established as a powerful tool for the analysis of genes leading to a wide spread of its application in research. Its basic qualities (sensitivity, specificity and quickness) also offer facilities for its application to the detection and amplification of foreign and aberrant genes. Recent reports have already shown its ability to detect microorganisms and to identify genes related to molecular diseases. Therefore, its application to the diagnosis of infectious and genetic diseases is expected in the next future. However, some problems have to be solved before the use of this technology is routine in clinical chemistry.

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.

Mapping of six epitopes on haemocyanin subunit Aa 6 by immunoelectron microscopy.

Monoclonal antibodies directed against the haemocyanin of the scorpion Androctonus australis were raised in order to map antigenic determinants (epitopes). The method of mapping employed in this study is molecular immunoelectron microscopy. It consists of a direct electron microscopic observation of antigenic molecules labelled with monoclonal antibodies. The epitopes are then localized in a small region of the external surface of the antigenic molecule whose architecture and quaternary structure are well known. Six monoclonal antibodies have been selected and epitopes have been circumscribed within a small area of one subunit among the 24 subunits composing the whole antigenic molecule.

Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing.

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy.

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)