Fully Detargeted Polyethylene Glycol-coated Adenovirus Vectors Are Potent Genetic Vaccines and Escape from Pre-existing Anti-adenovirus Antibodies

Abstract

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.

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

“…24,26,28 However, our data suggest that 20K-PEGylated Ad5, which showed the greatest reduction in immune responses and achieved the lowest level of cell association, in combination with compstatin, could represent a feasible avenue for further development of systemic adenoviral therapy. We want to emphasize that this study has again revealed the importance of using relevant models when studying immune responses against adenoviral vectors intended for use in humans.…”

“…This is in contrast to what has been observed in mice where PEGylation had considerable capacity to reduce immune responses and to enhance transgene expression. [24][25][26]28 We observed that the donors' ability to neutralize Ad5 had a considerable impact on the immune response both for unmodified and PEGylated Ad5. Interestingly, the presence of NAbs in plasma did not directly correlate with the neutralizing ability in whole blood, indicating that other mechanisms may be involved as well or that non-neutralizing antibodies, as determined in plasma samples, may be neutralizing in whole blood.…”

“…This is in agreement with previous observations, that high molecular weight PEG molecules have better ability to protect adenovirus in the circulation than low molecular weight PEG molecules. 28 It should be noted that complement activation and Ad5 binding to blood cells lead to rapid phagocytic clearance in the liver and spleen of mice. 23,40 Furthermore, splenectomy experiments have revealed that this organ is the major source for inflammatory cytokines.…”

“…26 On the basis of previous in vitro experiments it is believed that high molecular weight PEG molecules achieve more effective detargeting of Ad5 than low molecular weight PEG molecules. 27,28 In mice it has recently been shown that viruses coated with 20K-PEG molecules undergo less uptake in the liver compared with viruses coated with PEGs of lower molecular weight. 28,29 Wisse et al 30 propose that the physical size of the particle might be the determinant for infection.…”

“…27,28 In mice it has recently been shown that viruses coated with 20K-PEG molecules undergo less uptake in the liver compared with viruses coated with PEGs of lower molecular weight. 28,29 Wisse et al 30 propose that the physical size of the particle might be the determinant for infection. Adenoviruses with high molecular weight PEGs that are larger than the threshold of the liver sinusoidal fenestrae would be detargeted from the liver because of their size, not necessarily that the PEGylation is more efficient.…”