eduBSCI 3961Research Proposal: 3 credit hours BACKGROUNDAND HYPOTHESIS Staphylococcus aureus is now the most common invasive bacterial pathogen inchildren in the US, and new targets of intervention are urgently needed. Our research group has discovered that thetoxin LukAB is abundantly produced in the setting of invasive human infectionand is targeted by the host response. LukABis a highly conserved exotoxin that is critical to S.
aureus pathogenesis in both invitro and in vivo models. The Thomsen laboratoryhas collaborated with the Crowe Laboratory at Vanderbilt to isolate and purifyhuman monoclonal antibodies (mAbs) with LukAB-specific neutralizing activityfrom children with invasive S. aureusinfections.
In addition, they havepurified mAbs targeting important surface antigens. We hypothesize that acombination of mAbs that neutralize toxicity (anti-LukAB mAbs) as well asopsonize the bacterium for improved host killing (anti-surface mAbs) willpotently facilitate neutrophil killing of S.aureus in vitro. PROPOSEDSTUDY LukAB is secreted by S. aureus as a heterodimer, with oligomerization and pore formationoccurring on the surface of phagocytes at the time of cellular destruction. Our recent data indicate that neutralizing anti-LukAB human mAbsachieve toxin neutralization by distinct mechanisms. Based on these findings,we hypothesize that an oligoclonal mixture of anti-toxin mAbs with highaffinity and distinct properties will have increased potency in the blockage ofLukAB-mediated cytotoxicity. To assessthis, we propose a series of related experiments: In vitro toxin neutralization with distinctmAb combinations.
We willassess potentiation of activity (i.e., combination effect) in a series ofexperiments measuring mAb neutralization of LukAB-mediated neutrophil killing. Methods: Anti-LukAB mAb preparations (or isotypecontrols) will be incubated in the presence of LukAB for 30 minutes, prior tothe addition of neutrophil-like HL-60 cells.
After a one-hour intoxication,Cell Titer® Aqueous One dye will be added, and neutralization of cytotoxicitywill be measured as a function of the percentage of cells that remain alivefollowing intoxication relative to controls. Experiments will be performed intriplicate with cells from independent vials. We willdetermine the capacity of selected mAb combinations to protect against hostcell killing.
We hypothesize that a combination of antibodieswith diverse neutralizing mechanisms and Fc-mediated immune enhancing functionswill cooperate to facilitate maximal bacterial killing within human blood. 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. 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.