Toxoids of Pseudomonas aeruginosa toxin A: production by chemical and genetic means

Abstract

Several toxin A toxoids were produced by reacting purified toxin A with formalin or formalin-lysine. Treatment with formalin alone resulted in a 1,000-fold decrease in cytotoxicity of toxin A without adversely affecting enzymatic activity. The antigenicity was moderately altered, but immunogenicity appeared to be unaffected, as evidenced by the ability of the toxoid to elicit good titers of neutralizing antibody. However, on removal of formalin and after storage at 37 C, formol toxoid showed a partial reversion to toxicity. The addition of lysine to formalin-toxin A mixtures increased the rate and extent of detoxification and destroyed enzymatic activity. Although the formalin-lysine toxoid did not revert to toxicity, it displayed a reduced immunogenicity even when employed with an adjuvant. On the basis of these results, an alternative method was selected for detoxification of toxin A. A mutant (PAO-PR1), which produces a CRM protein (CRM 66), was isolated after chemical mutagenesis. CRM 66 has the same molecular weight as toxin A and is antigenically indistinguishable from it. The cytotoxicity and lethality to mice of CRM 66 was found to be greatly reduced in comparison to that of toxin A, a difference apparently due to a reduction in enzymatic activity.