Immunochemistry of scorpion alpha-toxins: purification and characterization of two functionally independent IgG populations raised against toxin II of Androctonus australis Hector.

We report the isolation and characterization of two IgG populations specific to two synthetic peptides corresponding to two antigenic sites of toxin II of the North African scorpion Androctonus australis Hector. Firstly, thanks to the use of: (1) antigenic homology studies between toxin II of A. australis Hector and toxin III of Buthus occitanus tunetanus, (2) chemical modification of toxin II of A. australis Hector, and (3) prediction of the localization of the four major antigenic sites of scorpion alpha-toxins by the method developed by Hopp and Woods [Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981)], we have established that the region around the disulfide bridge between cysteines 12 and 63 as well as the stretch of residues 50-59 probably each enclosed an antigenic site. Secondly, the synthetic replicates of these regions linked to Sepharose allowed us to isolate, by immunoaffinity chromatography, two IgG populations from the whole anti-toxin II of A. australis Hector IgGs. Finally, each of these two IgG populations was shown to be specific to one antigenic site as evidenced by the multideterminant effect on the slopes of binding curves developed by Berzofsky et al. [Biochemistry 15, 2113-2121 (1976)]. Furthermore, these two IgG populations were found to be functionally independent and this could be related to the fact that the two regions carrying the two antigenic sites are not close to each other in space and that there is neither steric hindrance nor cooperative effects between them. The association constant of these site-specific IgG populations was calculated and found to be equal to 1.18-5.14 X 10(9) l/mole for IgG anti-site 1 and 1.16-5.62 X 10(9) l/mole for IgG anti-site 2 respectively by Sips [J. chem. Phys. 16, 490-495 (1948)], Scatchard [Am. N.Y. Acad. Sci. 51, 660-772 (1949)] and Steward and Petty [Immunology 23, 881-887 (1972)] representations. The index of heterogeneity of 0.9 for anti-P1 and anti-P2 indicates the purification of essentially homogeneous affinity IgG populations.

Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

Immunogenicity and antigenicity of conserved peptides from the envelope of HIV-1 expressed at the surface of recombinant bacteria.

We expressed peptides from the HIV-1 envelope protein at the surface of Escherichia coli by genetic insertions into an exposed loop of the outer membrane protein LamB. Recombinant bacteria expressing eight peptides from gp110 (pep1-pep8), conserved between HIV-1 and HIV-2, were used as live immunogens in rabbits by the intravenous route. The eight constructions elicited anti-LamB antibodies, showing that the hybrid proteins were immunogenic. One of them, LamB-pep8, gave rise to antibodies able to react with gp160 and to neutralize HIV-1 in vitro. We also show that this type of recombinant E. coli can provide a convenient reagent to monitor and characterize specific antibodies. Recombinant clones were used to test sera of seropositive individuals, as well as to narrow down the monoclonal antibody 110-1 recognition site to a cluster of eight residues at the carboxy-terminal end of gp110.

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus.

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.

Human plasmatic apolipoprotein H binds human immunodeficiency virus type 1 and type 2 proteins.

Apolipoprotein H (apo H), isolated from human plasma albumin solution, was shown to capture HIV-1-related antigens from antigen-positive sera (HIV-1 AG+) of AIDS patients, by using HIV-1-specific polyclonal antibodies. In an enzyme-linked immunosorbent assay and ligand blot and dot assays, apo H was able to bind recombinant retroviral HIV antigens, especially Gag proteins p18 of HIV-1, p26 of HIV-2, and Env gp160 of HIV-1. Binding was shown to be pH and NaCl dependent, with an optimum at acidic pH and low ionic strength. Specificity was demonstrated by saturation of this binding and inhibition either by homologous competition or by specific antisera. Binding was also observed in cell line-harvested viral proteins. The mechanism of this apo H-polyspecific binding is discussed in relation to conformational changes due to the influence of lipids or detergents.

Purification of contracture-inducing insect toxins from Buthinae scorpion venoms by immunoaffinity and high pressure liquid chromatography.

A method of rapid and selective detection and purification of contracture-inducing insect toxins from Buthinae scorpion venoms is described in this paper. It consists of two main steps: the first one is specific as it uses immunoaffinity chromatography with antibodies against Androctonus australis Hector IT; the second, reverse phase high pressure liquid chromatography, allows the final separation of the different toxins present in each of the three venoms studied. Two, three and four insect toxins have been purified, respectively, from the venoms of Androctonus australis Hector, Buthus occitanus mardochei and Leiurus quinquestriatus quinquestriatus. This work demonstrates that, in Buthinae venoms, contracture-inducing insect toxins antigenically related to AaH IT, the first one purified, constitute the most important and, in some cases, the only toxins present.

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel.

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.

Immunochemical evidence for a role of complex carbohydrate chains in thyroglobulin antigenicity.

Thyroglobulin secreted by porcine thyroid cells in serum-free culture was previously found (Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293) to contain more highly branched complex carbohydrate chains than the thyroid-derived molecules. When assayed for their ability to react with polyclonal antibodies directed against the natural prohormone in competitive radioimmunoassays, using the porcine antigen as iodinated tracer and standard competitor, in vitro thyroglobulin appeared to be 4-fold less immunoreactive than the in vivo molecule. However, both types of thyroglobulin exhibited superimposable incomplete displacement curves after peripheral deglycosylation using a mixture of neuraminidase, alpha-, and beta-galactosidases while they behave as good competitors as the native antigen after removal of the majority of the carbohydrate chains by Endo-beta-N-acetylglucosaminidase F. Solid-phase binding assays revealed that in vitro thyroglobulin was able to bind less antibodies than its thyroid-derived counterpart before as well as after treatment with exo- or endoglycosidases. Furthermore, only 20% of thyroglobulin biosynthetically labeled with glucosamine could be precipitated with specific antibodies followed by addition of staphylococcal-protein A, whereas up to 61% of the labeling was specifically bound when the antibodies were preincubated with protein A. After pronase digestion, both thyroglobulin-like material displayed different carbohydrate structures as judged by concanavalin A-Sepharose analysis. Thus, substituting multiantennary complex carbohydrate chains to the usual biantennary and high mannose structures present on thyroid-derived thyroglobulin had a profound effect on the prohormone immunoreactivity.

Differential effects of defined chemical modifications on antigenic and pharmacological activities of scorpion alpha and beta toxins.

Specific chemical modifications of scorpion alpha and beta toxins have been used to study the involvement of particular residues in both the pharmacological and the antigenic sites of these toxins. Modification by 1,2-cyclohexanedione of arginine-27 of a beta toxin, Centruroides suffusus suffusus toxin II, drastically decrease the antigenic activity without any influence on the pharmacological activity. Conversely, modification by the same reagent of arginine-2 of an alpha toxin, Androctonus australis Hector toxin III, led to a 100-times less pharmacologically potent derivative and did not induce a significant loss of antigenic activity. Excision of the N-terminal pentapeptide of another alpha toxin, Buthus occitanus mardochei toxin III, by pepsin digestion led to a non-toxic derivative retaining full antigenic activity. Thus, the N-terminal part of the conserved hydrophobic surface of the toxin is highly implicated in the pharmacological activity, whereas the region of arginine-27, located in the alpha helix situated on the back surface, opposite the conserved hydrophobic region, is fully implicated in the antigenic activity and is far from the pharmacological site. These results are good arguments in favor of the idea that in scorpion toxins the surfaces implicated in the pharmacological and the antigenic activities do not overlap. Since the antigenic sites are present in highly variable sequence the development of an efficient polyvalent serotherapy is questionable.

Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin.

Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins.

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region.

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes.

CTL constitute an essential part of the immune response against the HIV. CTL recognize peptides derived from viral proteins together with the MHC class I molecules on the surface of infected cells. The CTL response could be important in prevention or control of infection with HIV by destroying virus-producing cells. In this study we have attempted to identify peptide epitopes recognized by HIV-specific CTL. Using synthetic peptides, we have identified six conserved peptidic epitopes on the gp120 envelope glycoprotein recognized by polyclonal human CTL in association with HLA-A2 class I transplantation Ag. These results were confirmed by two approaches: i) blocking of CTL activities with antibodies specific for three of these conserved peptides; and ii) construction of doubly transfected P815-A2 target cells, using deletions of the HIV env gene. Vaccination or immunotherapy in HLA-A2 individuals can thus be considered using highly conserved HIV env peptidic sequences.

Induction of anti-human immunodeficiency virus (HIV) neutralizing antibodies in rabbits immunized with recombinant HIV--hepatitis B surface antigen particles.

Fragments of the human immunodeficiency virus (HIV) envelope coding region have been fused with the hepatitis B virus envelope middle protein. In this system, HIV antigenic determinants are exposed at the surface of a highly antigenic structure, the hepatitis B surface antigen particle. Immunization of rabbits with these particles elicited antibodies directed against both parts of the hybrid protein. One of the rabbit antisera not only exhibited a neutralizing effect on the original HIV1 isolate but also on a divergent Zairian isolate. The HIV sequence in this recombinant is 84 amino acids long and contains conserved and variable domains and a region critical for interaction with the CD4 receptor. Such recombinant antigens could be primary elements in the design of a polyvalent vaccine.