Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product-specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.
Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously
accumulate in the liver as the result of their direct binding to blood
coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex
with heparan sulfate proteoglycan (HSPG) at the surface of liver cells.
Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver
transduction efficiencies 4-logs lower than HAdV5, even though both vectors
carry the same hexon capsomeres. In order to reconcile this apparent paradox, we
investigated the possible role of other viral capsid proteins on the
FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and
CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound
to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its
Gla-domain, and enhanced the binding of both vectors to surface-immobilized
hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the
positive effect of FX on HAdV5 binding to HSPG and cell transduction did not
depend on the penton base RGD and fiber shaft KKTK motifs. However, we found
that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but
a negative effect which did not involve the cell attachment or endocytic step,
but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex.
By cellular imaging, HAdV5F35 particles were observed to accumulate in the late
endosomal compartment, and were released in significant amounts into the
extracellular medium via exocytosis. We showed that the stability of serotype 5
hexon∶FX interaction was higher at low pH compared to neutral pH, which
could account for the retention of FX-HAdV5F35 complexes in the late endosomes.
Our results suggested that, despite the high affinity interaction of hexon
capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the
dominant determinant of the internalization and trafficking pathway of
HAdV5-based vectors.
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