CD1 and iNKT cells mediate immune responses against the GBS hemolytic lipid toxin induced by a non-toxic analog

Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known as R-P4 had reduced bacterial dissemination during systemic infection. However, mechanisms important for R-P4 mediated immune protection was not understood. Here, we show that immune serum from R-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naive mice from GBS infection. Further, CD4(+) T cells isolated from R-P4-immunized mice proliferated in response to R-P4 stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations, R-P4 immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells from R-P4 vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins. Author summaryAlthough many pathogens encode lipid toxins that contribute to disease pathogenesis, strategies to neutralize these toxins are lacking. The hemolytic lipid toxin encoded by Group B Streptococcus (GBS) is a major virulence factor that promotes disease including preterm births, in utero fetal injury and morbidity of human neonates and adults. No strategies exist to mitigate the effects of the GBS toxin, and neutralizing antibodies have not been isolated from infected patients. The global burden of GBS disease remains high with approximately 4 million neonatal deaths occurring worldwide each year and increasing rates of infections occur in adults. Using chemical synthesis, we synthesized a derivative of GBS toxin known as R-P4. We show that vaccination with this non-toxic analog induces the production of antibodies that promotes GBS killing, confers protection to naive mice, neutralizes the cytotoxic activity and diminishes GBS infection during pregnancy. Furthermore, we show that lipid analog mediated protection requires antigen presentation by host molecules such as CD1d and immune responses of CD1d restricted T cells such as iNKT cells. These results provide the foundation for the design of novel therapeutic strategies that target lipid cytotoxins.Despite observations that lipid toxins continue to increase the severity and rate of infections caused by pathogens such as GBS, Mycobacteria and Pseudomonas, strategies to neutralize these toxins remain undefined. Our results indicate that a nontoxic analog of one such toxin can induce protective immunity during systemic and pregnancy associated infection, through lipid antigen presentation which provides the foundation for promising future strategies to neutralize the effect of lipid toxins during human infections.

Automated summary

By investigating the R-P4 compound, a non-toxic analog of the GBS lipid toxin, it was discovered that immunizing mice with this compound could promote opsonophagocytic killing of the bacteria and protect the mice from GBS infection. CD4(+) T cells from R-P4-immunized mice showed increased proliferation in response to R-P4 stimulation, and this immune response was CD1d- and iNKT cell-dependent. Transferring iNKT cells from R-P4-vaccinated mice to other mice significantly reduced GBS dissemination, and maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy.