Determination of particle heterogeneity and stability of recombinant adenovirus by analytical ultracentrifugation in CsCl gradients.

Recombinant adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles. To determine particle heterogeneity we utilized analytical ultracentrifugation (AUC) in CsCl density gradients. Preparations of three different rAd vectors were assessed. AUC was able to resolve multiple density forms including two empty capsid types in various virus preparations. One unusual density form (form V), was noninfectious and lacked protein VI. AUC was able to quantify empty capsids and monitor their removal during process development. Their relative concentrations were reduced by either addition of an immobilized zinc affinity chromatography (IZAC) step or by extension of the infection time. The Adenovirus Reference Material (ARM), a wild-type Ad5, had 2.2% empty capsids and no other detectable minor particle forms. Finally, AUC was utilized to monitor the thermal instability of the three rAd vectors via the transformations of different density forms. The vector and empty capsids containing protein IX were more stable than those without IX. Together, these results exemplify AUC in CsCl density gradients as a valuable technique for evaluating product particle heterogeneity and stability.

Investigations of PEGylated recombinant adenovirus, using fluorescein-labeled polyethylene glycol.

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.

Comparative thermal stabilities of recombinant adenoviruses and hexon protein.

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T1, T2 and T3) occurring between 25 degrees C and 90 degrees C. T1, which occurred at 46.7 degrees C in vPBS and 49.4 degrees C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 degrees C and 78 degrees C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 degrees C (T1), 70.5 degrees C (T2A), 73.6 degrees C (T2B), and 77.4 degrees C (T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 degrees C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.

Characterization of empty capsids from a conditionally replicating adenovirus for gene therapy.

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.

Complement component C1q and anti-hexon antibody mediate adenovirus infection of a CAR-negative cell line.

Realization of the potential clinical utility of recombinant adenovirus for gene therapy or vaccine development depends on a better understanding of the role of naturally occurring or therapy-induced anti-adenovirus antibodies. This study addresses the impact of anti-adenovirus neutralizing antibodies and the complement protein C1q on adenovirus infection of coxsackie and adenovirus receptor (CAR)-positive, and especially CAR-negative cells. Initially, transduction efficiency of adenovirus vectors was assessed in the presence or absence of human sera derived from healthy individuals that were seropositive for anti-adenovirus neutralizing antibodies. Infection was monitored by transgene expression in vitro using a replication-deficient adenovirus encoding green fluorescent protein (Ad-GFP). HeLa cells (CAR-positive) were readily infected by Ad-GFP and increasing concentrations of pooled sera increasingly inhibited infection. In contrast, rhabdomyosarcoma (RD) cells, a CAR-negative cell, were poorly infected by Ad-GFP. However, in the presence of human serum, robust GFP expression was observed. This expression was completely abrogated if the human serum was heat-inactivated. Addition of purified human C1q protein to the heat-inactivated serum restored GFP expression. Similar results were seen when human C1q protein was added to purified anti-hexon antibodies, but not to anti-fiber or anti-penton base antibodies, thus implicating anti-hexon antibodies as the infective antibody component of the human sera. These studies suggest that complement protein C1q and anti-hexon antibodies together can mediate efficient adenovirus infection in CAR-negative cell types.

Characterization of hemodynamic events following intravascular infusion of recombinant adenovirus reveals possible solutions for mitigating cardiovascular responses.

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.