Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

Abstract

The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton–αvintegrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1 + sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting

“…In contrast, clodronate barely reduced resident macrophages in lung, small bowel, heart, and kidney (data not shown).Clodronate increased Ad.MBP.CMV EC lung expression by 2-fold but did not significantly alter EC transgene expression level or the EC-specific expression pattern in liver, spleen, heart, kidney, muscle, pancreas, small bowel, or brain (Figure 4B). The lack of increase in hepatocyte was surprising given prior reports on the scavenging function of liver Kupffer cells 25 , however, others have also reported a modest, though statistically insignificant level of clodronate-mediated Ad vector liver expression enhancement 26 . Most importantly our data demonstrate that circulating monocytes and macrophages were dispensable for Ad.MBP.CMV organ EC expression.…”

Ad5:Ad48 Hexon Hypervariable Region Substitutions Lead to Toxicity and Increased Inflammatory Responses Following Intravenous Delivery

“…We have previously shown that ER-TR7+ reticular fibroblasts and MAdCAM-1+ sinus-lining endothelial cells, both structural components of the splenic micro-architecture, are unaffected by pre-treatment with clodronate liposomes [5]. Interestingly, we have demonstrated that these cell are responsible for a large proportion of viral transgene expression at late time-points in macrophage-depleted animals [5, 32]. Therefore, we investigated whether these cells were responsible for accumulating viral particles 1h post-injection (Fig.3d).…”

Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5) Vectors Display Reduced Hepatotoxicity but Induce T Lymphocyte Phenotypes Similar to Ad5 Vectors

“…We initiated studies by assessing the systemic cytokine and chemokine responses in two strains of mice after vaccination, inbred C57BL/6 mice that had been extensively utilized for characterizing Ad vector immunology, and outbred MF1 mice that had been used in previous studies assessing Ad vector toxicity (4,8,12,14,19,21). The mice (C57BL/6 or MF1; n ϭ 3 to 4/group) were inoculated intravenously (i.v.)…”

“…Ad5 vectors at high doses also exhibit hepatotoxicity as a result of liver tropism due to the binding of blood coagulation factor X (FX) in the systemic circulation (7)(8)(9)(10). Both of these properties appear to be mediated primarily by the hexon hypervariable regions (HVRs) (8,(11)(12)(13)(14)(15)(16). HVRs are highly variable among Ad serotypes and represent the primary determinant of neutralization specificity (17)(18)(19)(20).…”