Influence of chimeric human-bovine fibers on adenoviral uptake by liver cells and the antiviral immune response.

Human adenoviruses (HAdV) are widely used for in vitro and in vivo gene transfer. Viral hepatotropism, inflammatory responses and neutralization by pre-existing antibodies (NAbs) are obstacles for clinical applications of HAdV vectors. Although the multifactorial events leading to innate HAdV toxicity are far from being elucidated, there is a consensus that the majority of intravenously injected-HAdV vectors is sequestered by Kuppfer cells, probably independently of coagulation factors. In this study, we show that the adenoviral-associated humoral and innate cytokine immune responses are significantly reduced when HAdV-5 vector carrying human bovine chimeric fibers (HAdV-5-F2/BAdV-4) is intravenously injected into mice. Fiber pseudotyping modified its interaction with blood coagulation factors, as FIX and FX no longer mediate the infection of liver cells by HAdV-5-F2/BAdV-4. As a consequence, at early time points post-infection, several cytokines and chemokines (IFN-gamma, IL-6, IP-10, MCP-1, RANTES and MP1beta) were found to be present at lower levels in the plasma of mice that had been intravenously injected with HAdV-5-F2/BAdV-4 compared with mice injected with the parental vector HAdV-5. Moreover, genetic modification of the fiber allowed HAdV-5-F2/BAdV-4 to partially escape neutralization by NAbs.

Non-heparan sulfate GAG-dependent infection of cells using an adenoviral vector with a chimeric fiber conserving its KKTK motif.

Recently, the potential involvement of the putative heparan sulfate proteoglycans (HSPG) binding motif, KKTK, in mediating HAdV-5 liver cell infection following intravascular virus delivery has been debated. In the present study, we demonstrated that HSPGs were not involved in the in vitro infection process of an adenoviral vector harboring chimeric fibers without mutation in the KKTK motif, HAdV-5-F2/BAdV-4. The entry of HAdV-5-F2/BAdV-4 into cells occurs by two mechanisms 1) the attachment of HAdV-5-F2/BAdV-4 to the surface of cells requires N-glycosylation, 2) the uptake of the virus is effective after interaction with a co-receptor, putatively the chondroitin sulfate C. Together, these results contribute to improving our understanding of the molecular mechanisms determining HAdV's infectivity in vitro and may aid in designing novel HAdV-based vectors for gene therapy applications.

Efficient species C HAdV infectivity in plasmocytic cell lines using a clathrin-independent lipid raft/caveola endocytic route.

Hematopoietic cells are known to be refractory to species C human adenovirus (HAdV) infection; however, the reason for this has not been clearly established. We have previously demonstrated that this nonpermissivity is the consequence of inefficient HAdV particle uptake, notably in B lymphocytes. We noted that while the protein clathrin is observed in association with membranes in epithelial cells, it is found predominantly in the cytoplasm of hematopoietic cell lines. So it appears that altered clathrin-coated pit endocytosis could explain the weak HAdV uptake in B cells. In contrast, mature B cell plasmocytes are permissive to HAdV. However, this is not the result of clathrin-coated pit endocytosis since this process is also inefficient in these cells. Confocal microscopy showed colocalization between HAdV particles and caveolae/lipid rafts in plasmocytes. Moreover, inhibiting caveola endocytosis by depletion of cholesterol or expression of dominant negative caveolin-1 in these cells results in a 50-70% reduction in HAdV infectivity. It appears that caveola endocytosis and nonclathrin noncaveola endocytosis are used by HAdV to enter plasmocytes in response to a loss of the clathrin-dependent pathway. Thus targeting of caveolae by modifying the capsid of HAdV may represent an alternative approach to enhancing uptake in most hematopoietic cells for future gene therapy.