Abstract
Intravascular (iv) delivery of adenovirus (Ad) vectors is hindered by acute toxicity that is triggered by trapping of virus in the reticuloendothelial system (RES) within minutes of administration, and subsequent production of pro-inflammatory cytokines/chemokines. While Ad localizes to tissue macrophages and endothelial cells of the liver, spleen and lung within minutes of iv delivery, the mechanism of this reticuloendothelial targeting is unknown. We have identified a novel pathway by which platelets mediate trapping of Ad in the RES. Rapidly after iv administration fiber independent interaction between Ad and circulating platelets can be seen by Southern blot, by transmission electron microscopy (TEM) of isolated platelets and blood cell fractionation of mice receiving radiolabeled Ad. Blood platelet levels drop after Ad administration, while serum levels of the platelet activation marker soluble CD62p rise. Within 5 minutes of administration, Ad and platelets co-localize in the liver sinusoids along with endothelial and Kupffer cells. TEM showed Ad particles inside degranulated platelets and platelet aggregates within the liver sinusoids. Ad particles could also be seen inside Kupffer cells. Immunohistochemistry (IHC) showed widespread co-labelling of platelets (CD41), Kupffer cells (F4/80) and Ad (hexon) throughout the liver. In the spleen, Ad genomes were found within 5 minutes and Ad particles were detected in CD41 and F4/80 rich regions of the marginal zone by IHC. In the lung, Ad genomes were found within 5 minutes. Platelet accumulation was seen by IHC after delivery of Ad vectors, with platelet-endothelial interactions visible in the pulmonary vasculature by TEM. To determine the contribution of Ad-platelet interactions towards toxicity, mice were pre-injected with a platelet depleting antibody prior to virus delivery. At 6 hours post virus delivery, vector genomes in liver, lung and spleen were detected by Southern blot, and circulating serum cytokine levels were determined. Serum levels of the pro-inflammatory cytokines/chemokines IFN-|[gamma]|, IL-6 and MCP- 1 were lower in platelet depleted mice. The effect of Ad neutralizing antibodies on Ad-platelet interactions was also determined in mice that had been pre-immunized with the same vector prior to iv Ad vector administration. Early after vector administration significantly lower levels of Ad genomes were associated with circulating platelets in pre-immunized mice. While the levels of Ad deposition in the liver were unchanged in pre-immunized mice, there was reduced Ad deposition within the lungs of pre-immunized mice. Taken together these data suggest that rapid interaction between Ad and platelets occurs after iv administration. Platelet-Ad interactions apparently play a role in RES sequestration and degradation of Ad, and subsequent toxicity. We are currently attempting to identify the structural components involved in Ad-platelet interactions in vivo and investigating the contribution of this interaction to sequestration, degradation and toxicity, after Ad administration.
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