North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.
We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.
Olvera
Alejandro
A
Departamento de Medicina Molecular y Bioprocesos. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico.
Ramos-Cerrillo
Blanca
B
Estévez
Judith
J
Clement
Herlinda
H
de Roodt
Adolfo
A
Paniagua-Solís
Jorge
J
Vázquez
Hilda
H
Zavaleta
Alfonso
A
Arruz
María Salas
MS
Stock
Roberto P
RP
Alagón
Alejandro
A
D000595:Amino Acid Sequence; D000818:Animals; D000997:Antivenins / Q000276:immunology*; D001483:Base Sequence; D003429:Cross Reactions; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D013111:Spider Venoms / Q000276:immunology*
D000997:Antivenins; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D