Ex vivo infection assay.  Maintaining thehypothesis that a combination of antibodies with diverse neutralizingmechanisms and Fc-mediated functions will cooperate to facilitate maximalbacterial killing, we will assess mAb combinations in the presence ofneutrophils plus complement. Clinical isolates of S. aureus obtained from the Thomsen Lab cohort of invasivelyinfected children will be incubated with neutrophils at a multiplicity ofinfection (MOI) of 10, diluted guinea pig complement, and mAb combinations atselected doses.

CFU and neutrophil viability (via LDH release assay) will beassessed after 90 minutes of infection. The use of active and heat-inactivatedcomplement will allow the determination of the complement-mediated contributionto phagocytosis. The most potent mAb combinations for CFU reduction andneutrophil protection will provide insight into the critical components of thehost-leukocidin interaction.  Selectedcombinations from the above experiments will then be assessed in future studiesin the Thomsen Lab, including a whole blood killing assay to further evaluatethe hypothesis that a combination of antibodies with distinct neutralizingmechanisms and Fc-mediated, immune enhancing functions will cooperate tofacilitate maximal bacterial killing within human blood.  Together, these experiments will test animportant hypothesis and provide insights into host-pathogen interactions.

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  The most potent mAb combinations identifiedin these studies will represent a potential modality for intervention against S. aureus infection in humans, whilealso (by assessment of the distinct properties of that combination) providingnovel insights into the critical components of the host-toxin interaction.