An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood

Abstract

Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37 1C for up to 8 h. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

“…29 However, studies on Ad5 interaction in human blood indicate that binding to erythrocytes has a greater role than interaction with blood coagulation factors. 24,30 Moreover, data from clinical trials strongly indicate that systemic administration of Ad5 do not affect the liver to the same extent as has been observed in mouse models. 30,31 Treatment with oncolytic Ads has been well tolerated and more importantly no significant liver toxicity has been reported to date after Ad infusions including numerous injections directly into the hepatic artery.…”

“…22,23 Therefore, we evaluated the recombinant GFP-expressing, fiber-modified Ad5 vectors in a newly developed human blood loop system. 24 Viruses were mixed with whole blood or medium and incubated in heparin-coated polyvinyl chloride tubing at 37 1C while rotating. We used blood from six healthy donors (D); four with low ability to neutralize Ad5 (D1, D2, D5 and D6) and two with high neutralizing ability (D3 and D4), to study the degree of neutralization in whole blood by adding the virus/blood sample dilutions to 911 cells and measuring GFP expression the day after.…”

“…The ex vivo blood loop model for whole human blood has been described earlier. 24 Minor changes are that 0.5 U ml ml -1 of hirudin (LEO Pharma Nordic, Ballerup, Denmark) was immediately added to the venous blood in heparinized Falcon tubes. The blood was then transferred to heparin-coated polyvinyl chloride tubing, 21 cm long with an inner diameter of 4 mm (Corline, Uppsala, Sweden).…”

Human Coagulation Factor X-Adenovirus Type 5 Complexes Poorly Stimulate an Innate Immune Response in Human Mononuclear Phagocytes

“…Additionally, the data suggested that hFX binding to HAd5 did not interfere with polyclonal anti-Ad IgG binding. In human blood, elevated levels of proinflammatory cytokines can be detected within hours ex vivo (36) and in vivo (37) after systemic administration. While the TLR4 internalization is thought to be a requirement for the induction of interferon-␤ (IFN-␤) (35,38) through a TRIF/TRAF3/IRF3 pathway, the possibility of TLR4 signaling occurring directly from the cell surface and induction of proinflammatory cytokines via MyD88-NF-B cannot be formally excluded.…”

Adenovirus with Hexon Tat-Protein Transduction Domain Modification Exhibits Increased Therapeutic Effect in Experimental Neuroblastoma and Neuroendocrine Tumors

“…The DNA copy number was detected by using the primers Ad.Titration.F and Ad.Titration.R targeting the adenovirus E4 ORF1 region. Plasmid, pCR2.1(AdE4orf1), containing the same amplicon, was used for the generation of standard curve (10). Virus titer was calculated as the mean of three independent experiments.…”