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As with certain successful protein drug treatments, the attachment of polyethylene glycol (PEG) molecules to recombinant adenovirus (rAd) can augment their therapeutic potential. Unlike these proteins, the rAd particle has thousands of target sites for PEG conjugation. The reliable measurement of the average number of PEG molecules attached to the virion, or the degree of PEGylation (DP), is crucial not only for the characterization of PEGylated virus but also for optimization of the PEGylation reaction. Using a fluorescein-labeled PEG-SPA linker (SPA, succinimidyl ester of PEG propionic acid) with a 5-kDa linear PEG moiety, multiple preparations of fluoro-PEG-rAds were produced under various reaction conditions, purified, and analyzed by size-exclusion high-performance liquid chromatography (HPLC) with fluorescence quantification of the virus peak. The DP was strongly dependent on the percent linker concentration in the reaction. For example, under one set of conditions, fluoro-PEG-rAd samples prepared at 1.3, 2.5, 5.0, 7.4, and 10.0% linker concentration had DPs of approximately 540, 1,000, 1,590, 1,990, and 2,170, respectively. The fluoro-PEG-rAds were compared with a set of nonfluorescent PEG-rAds. Analytical ultracentrifugation in CsCl density gradients showed distinct peaks at decreased buoyant density corresponding to the increased DP of the rAd samples; sodium dodecyl sulfate-polyacrylamide gel electrophoresis/scanning densitometry showed decreased hexon monomer and penton base. Both techniques were used to estimate the DP of nonfluorescent PEG-rAds versus fluoro-PEG-rAds, and anion-exchange HPLC revealed the different surface chemistries of the two vector types. In summary, these studies should provide investigators with the ability to reproducibly prepare and characterize PEGylated rAds or other large viral or nonviral particles for further in vivo studies.

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.

The clinical application of recombinant adenoviruses as vectors for gene therapy brings about the need to develop new analytical methodologies for monitoring the quality of the viral production as well as establishing structure–function relationships. A mass spectrometry-based assay has been developed for the characterization of structural proteins of the recombinant adenovirus type 5 vector encoding human p53 tumor suppressor gene. The fingerprinting of the viral proteome was accomplished by integration of MALDI-MS and/or MALDI-PSD-MS with SDS–PAGE and RP-HPLC, followed by database search using MS-Fit and MS-Tag algorithms. Viral proteins (molecular weights ∼10,000–100,000 Da) corresponding to more than 95% of total protein mass were resolved and identified, which include hexon (II), penton base (III), peripentonal hexon-associated protein (IIIa), minor core protein (V), major core protein (VII), and other hexon-associated proteins (VI and VIII). An important finding of our studies was the identification of some precursor proteins (i.e., pVIII) and propeptides of precursor proteins (pVIII, pX, and pVI) present in the adenovirus sample. The information obtained allows direct and accurate assessment of the quality of recombinant adenoviruses.

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.

For a previously studied replication-deficient rAd, we found that the empty capsids in these preparations had a high amount of pVIII (precursor to adenovirus protein VIII). This precursor protein was quantified by SDS-PAGE densitometry or analytical RP-HPLC. These techniques determined the concentration of empty capsids (ECpVIII) in these preparations. But these techniques were not useful for quantification of the empty capsids from a conditionally replicating rAd due to low levels of pVIII. Here we studied alternative methods to quantify this different type of empty capsid.