EPI-peptide designer: a tool for designing peptide ligand libraries based on epitope-paratope interactions.

MOTIVATION: Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. RESULTS: To identify the interacting residues in a given antibody-antigen (Ab-Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab-Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. AVAILABILITY AND IMPLEMENTATION: All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ CONTACT: liza@icb.ufmg.br SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Cutaneous loxoscelism caused by Loxosceles similis venom and neutralization capacity of its specific antivenom.

Members of the spider genus Loxosceles pose a marked health risk to humans because of the seriousness of the necrotic and systemic effects of their bite, known as loxoscelism. The recent confirmation of Loxosceles similis in residences of Belo Horizonte in Minas Gerais Province, Brazil increases the local potential risk of loxoscelism at higher levels. The first characterization of the venom from this species showed that its main biological effects had a similar intensity as other species (e.g. Loxosceles intermedia, Loxosceles laeta, and Loxosceles gaucho). Therefore, we wished to further analyse the biological activity of the L. similis venom as well as the capacity of anti-L. similis-venom serum to reduce dermonecrotic effects to rabbit skin. Histological analysis of rabbit skin 2, 4 and 8h after intradermal injection of L. similis venom demonstrated a dense inflammatory infiltrate, edema, degeneration and necrosis of the skin muscle, dissociation of collagen fibers, and disruption of reticular fibers. Importantly, pre-incubation of the venom with anti-L. similis-venom serum significantly decreased all of these effects. Anti-L. similis antivenom generated antibodies that were strongly reactive to L. similis venom and capable of neutralizing the dermonecrotic effects in rabbits caused by this venom. Moreover, the antivenom significantly reduced the sphingomyelinase activity of L. similis crude venom. Venoms produced by male and female spiders were equally reactive towards anti-L. similis and anti-L. intermedia antivenoms, but female venom induced larger lesions on rabbits. In contrast, female venom acted as an immunization enhancer and protected animals from L. similis envenomation to a greater degree than male venom. In conclusion, the results shown in this study for L. similis antivenom merits a more in depth study of its properties, which may become a valuable tool against loxoscelism.

Protection against the toxic effects of Loxosceles intermedia spider venom elicited by mimotope peptides.

The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.

A protective immune response against lethal, dermonecrotic and hemorrhagic effects of Loxosceles intermedia venom elicited by a 27-residue peptide.

Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.