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) constitutes a biologically active domain. A critical residue appears to be histidine 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 syndrome. 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 shock. Antibodies raised in rabbits by immunization with the purified H135A 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 cells equally well as did TSST-1-specific polyclonal and monoclonal antibodies. In addition, the H135A antibodies blocked the production of tumor necrosis factor by spleen cells stimulated with rTSST-1. The toxicities 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, no elevation in the serum levels of interleukin-2 or tumor necrosis factor was measurable. Passive immunization with H135A antibodies also protected MRL-lpr/lpr mice against lethal challenge with rTSST-1. Finally, rabbits actively immunized with purified H135A did not succumb to infection with a transformed strain of Staphylococcus aureus expressing rTSST-1. Additional animal studies will be required to confirm the immunizing potential of H135A and the efficacy of H135A antibodies as a neutralizing antitoxin.