SpyCatcher-SpyTagged ApxIA Toxoid and the Immune-Modulating Yeast Ghost Shells

Actinobacillus pleuropneumoniaesecretes the hemolytic cytotoxins ApxI, ApxII, ApxIII, and ApxIV, which cause pleurop- neumonia in swine. Of these, ApxI is the most toxic. ApxIA, a repeats-in-toxin toxin-like protein, has strong hemolytic and cytotoxic activities. This study aimed to develop an immune modulator ApxIA toxoid, with a Spytag/Spycatcher pair (SC::ST pair), in yeast ghost shells (YGSs). These YGSs were utilized as ApxIA toxoid delivery platforms for -glucan components that can be recognized by the innate immune system. The SC::ST pair was used to conjugate the ApxIA toxoid to YGSs. The YGS-SC::ST-ToxApxIA was successfully phagocytosed by RAW 264.7 macrophages cells, without any toxicity. Further investigation revealed that YGS-SC::ST-ToxApxIA led to defective immune responses, in addition to increased levels of cytokines IL-1β, TNF-α, and IL-10. A membrane proteomic analysis, to determine preferential major histocompatibility complex binding of ApxIA-derived peptides, was performed and four ApxIA peptides were successfully identified by liquid chromatography with tandem mass spectrometry analysis. The identified peptides may serve as poten- tial vaccine candidates in immunobiology studies of A. pleuropneumoniae. Our results indicate that YGS-SC::ST-ToxApxIA can prevent A. pleuropneumoniae pleuropneumonia (APP) by inducing both humoral and cellular immune responses.