Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

As virus vectors for gene therapy approach the goal of successful clinical treatment, it is increasingly necessary for the product to be fully characterized. Empty capsids are perhaps the main extraneous component of recombinant adenovirus (rAd) products that are purified by column chromatography. Two diverse rAd products, one a replication-defective rAd and the other a conditionally replicating rAd, show different protein compositions of their empty capsids. The empty capsid type from the replication-defective rAd carrying the gene for p53 was previously determined to have approximately 1400 copies per particle of pVIII, the precursor to the hexon-associated protein VIII (Vellekamp et al., Hum. Gene Ther. 2001;12:1923-1936). Quantification of this protein is a useful measure of the amount of empty capsids in preparations of this vector. Here we purify and characterize empty capsids from the conditionally replicating rAd. This empty capsid type lacks any appreciable amount of pVIII but contains pVI and multiple forms of the L1 52/55K protein, mostly as disulfidelinked oligomers. Empty capsid from conditionally replicating rAd present new challenges in terms of its quantification, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis densitometry analysis suggests that the amount of this empty capsid in a preparation, like that of rAd p53 empty capsid, declines with increased time of infection. This empty capsid demonstrates heterogeneity by anion-exchange chromatography, electron microscopy, and CsCl density gradient centrifugation.

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Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.