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Highly purified adenovirus preparations are essential for fundamental research and therapeutic use. However, the impact of viral concentration during CsCl gradient ultracentrifugation on virion integrity is poorly understood, and this incomplete mechanistic understanding may adversely affect product quality and constrain further advances in research. In this study, we examined how viral input affects adenovirus infectivity, cytotoxicity, yield, and structural integrity using transmission electron microscopy and analysis tools. High-concentration preparations (AdV-HC) showed significant aggregation, capsid damage, and considerably lower TCID₅₀ values, while optimally concentrated preparations (AdV-OC) preserved intact icosahedral structures and high infectivity. Efforts to reverse aggregation failed, showing that excessive viral concentration during purification causes irreversible structural harm. These findings indicate that overloading during CsCl ultracentrifugation promotes vector aggregation, thereby compromising virion stability and biological activity. To preserve structural integrity and ensure consistent performance, we recommend maintaining the final purified vector concentration at or below 2 mg/mL. Based on this threshold, we defined theoretical optimal optical density (OD) ranges for adenovirus concentration in cell lysates prior to ultracentrifugation to minimize vector aggregation during purification. Therefore, this study provides a simple and effective strategy for improving the consistency, purity, and infectivity of adenovirus preparations for both research and clinical applications.

The usefulness for cancer therapy of replication-competent adenoviral vectors expressing therapeutic genes from the E3 region has been evaluated, but few reports have described replication-competent adenoviruses with insertions at the E1 region in the full viral genome. We investigated in different prostate cancer cells the oncolytic efficacy of the replication-competent adenovirus 11p vectors expressing adenovirus death (RCAd11pADP) and red fluorescence (RCAd11pRFP) proteins from the upstream E1 region. ADP/RFP gene expression was 2-3 logs higher in PC3 and DU145 cells than in LNCaP and RWPE-1 cells. E1A protein expression in PC3 and DU145 cells was notably increased after infection with the RCAd11pADP or RCAd11pRFP vector compared with the Ad11pwt virus. Toxicity assays revealed 2-5-fold greater oncolytic effects of RCAd11pADP compared to Ad11pwt. Although all three viruses suppressed subcutaneous PC3 tumour growth in nude mice, RCAd11pRFP had greater oncolytic effects than did the Ad11pwt virus, and RCAd11pADP exhibited significant anti-tumour effects via apoptosis in a xenograft model. Interestingly, the apoptosis triggered by RCAd11pADP was markedly enhanced in comparison to that by the vector expressing ADP from E3 region. Taken together, our findings suggest that RCAd11pADP can potentially be used for the treatment of prostate metastases in clinical settings.

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt; of the three, RCAd11pE3 was the most tolerant to heat treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47°C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability.