A MUTATION AT HISTIDINE RESIDUE-135 OF TOXIC-SHOCK-SYNDROME TOXIN YIELDS AN IMMUNOGENIC PROTEIN WITH MINIMAL TOXICITY

Structure-function studies have revealed that the region between amino acids 115 and 141 of toxic shock syndrome toxin 1 (TSST-1) constitute s a biologically active domain. A critical residue appears to be histi dine 135, since a site-directed mutation that alters the histidine to alanine (H135A) results in a loss of mitogenic activity and an absence of toxicity as measured in a rabbit infection model of toxic shock sy ndrome. We have characterized the mutant toxin further and report here on its immunogenic activity in rabbits and on the protective ability of mutant-specific antibodies in two animal models of toxin-mediated s hock. Antibodies raised in rabbits by immunization with the purified H 135A are fully cross-reactive with staphylococcal TSST-1 and wild-type recombinant TSST-1 (rTSST-1) expressed in Escherichia coli. The H135A antibodies neutralized the mitogenic activity for murine splenic T ce lls equally well as did TSST-1-specific polyclonal and monoclonal anti bodies. In addition, the H135A antibodies blocked the production of tu mor necrosis factor by spleen cells stimulated with rTSST-1. The toxic ities of rTSST-1 and H135A were compared in D-galactosamine (D-GalNH(2 ))-sensitized MRL-lpr/lpr mice. The nontoxicity of H135A was confirmed in this murine model of superantigen-induced septic shock No toxicity of H135A was demonstrable at doses of 60 mu g, while doses of rTSST-1 as low as 2 mu g caused significant mortality within 24 to 72 h after challenge. Furthermore, subsequent to challenge of mice with H135A, n o elevation in the serum levels of interleukin-2 or tumor necrosis fac tor was measurable. Passive immunization with H135A antibodies also pr otected MRL-lpr/lpr mice against lethal challenge with rTSST-1. Finall y, rabbits actively immunized with purified H135A did not succumb to i nfection with a transformed strain of Staphylococcus aureus expressing rTSST-1. Additional animal studies will be required to confirm the im munizing potential of H135A and the efficacy of H135A antibodies as a neutralizing antitoxin.