Research Proposal: 3 credit hours
Staphylococcus aureus is now the most common invasive bacterial pathogen in
children in the US, and new targets of intervention are urgently needed. Our research group has discovered that the
toxin LukAB is abundantly produced in the setting of invasive human infection
and is targeted by the host response.
is a highly conserved exotoxin that is critical to S. aureus pathogenesis in both in
vitro and in vivo models.
The Thomsen laboratory
has collaborated with the Crowe Laboratory at Vanderbilt to isolate and purify
human monoclonal antibodies (mAbs) with LukAB-specific neutralizing activity
from children with invasive S. aureus
infections. In addition, they have
purified mAbs targeting important surface antigens. We hypothesize that a
combination of mAbs that neutralize toxicity (anti-LukAB mAbs) as well as
opsonize the bacterium for improved host killing (anti-surface mAbs) will
potently facilitate neutrophil killing of S.
aureus in vitro.
LukAB is secreted by S. aureus as a heterodimer, with oligomerization and pore formation
occurring on the surface of phagocytes at the time of cellular destruction. Our recent data indicate that neutralizing anti-LukAB human mAbs
achieve toxin neutralization by distinct mechanisms. Based on these findings,
we hypothesize that an oligoclonal mixture of anti-toxin mAbs with high
affinity and distinct properties will have increased potency in the blockage of
LukAB-mediated cytotoxicity. To assess
this, we propose a series of related experiments:
In vitro toxin neutralization with distinct
mAb combinations. We will
assess potentiation of activity (i.e., combination effect) in a series of
experiments measuring mAb neutralization of LukAB-mediated neutrophil killing.
Methods: Anti-LukAB mAb preparations (or isotype
controls) will be incubated in the presence of LukAB for 30 minutes, prior to
the addition of neutrophil-like HL-60 cells. After a one-hour intoxication,
Cell Titer® Aqueous One dye will be added, and neutralization of cytotoxicity
will be measured as a function of the percentage of cells that remain alive
following intoxication relative to controls. Experiments will be performed in
triplicate with cells from independent vials.
determine the capacity of selected mAb combinations to protect against host
cell killing. We hypothesize that a combination of antibodies
with diverse neutralizing mechanisms and Fc-mediated immune enhancing functions
will cooperate to facilitate maximal bacterial killing within human blood.
Ex vivo infection assay. Maintaining the
hypothesis that a combination of antibodies with diverse neutralizing
mechanisms and Fc-mediated functions will cooperate to facilitate maximal
bacterial killing, we will assess mAb combinations in the presence of
neutrophils plus complement. Clinical isolates of S. aureus obtained from the Thomsen Lab cohort of invasively
infected children will be incubated with neutrophils at a multiplicity of
infection (MOI) of 10, diluted guinea pig complement, and mAb combinations at
selected doses. CFU and neutrophil viability (via LDH release assay) will be
assessed after 90 minutes of infection. The use of active and heat-inactivated
complement will allow the determination of the complement-mediated contribution
to phagocytosis. The most potent mAb combinations for CFU reduction and
neutrophil protection will provide insight into the critical components of the
combinations from the above experiments will then be assessed in future studies
in the Thomsen Lab, including a whole blood killing assay to further evaluate
the hypothesis that a combination of antibodies with distinct neutralizing
mechanisms and Fc-mediated, immune enhancing functions will cooperate to
facilitate maximal bacterial killing within human blood. Together, these experiments will test an
important hypothesis and provide insights into host-pathogen interactions. The most potent mAb combinations identified
in these studies will represent a potential modality for intervention against S. aureus infection in humans, while
also (by assessment of the distinct properties of that combination) providing
novel insights into the critical components of the host-toxin interaction.