Adenovirus vectors (Ad) are the second most frequently used vectors in clinical trials in the US to treat numerous inborn and acquired human diseases, including cancer. Although no cure so far is found for disseminated metastatic tumor disease, it is currently accepted that disseminated metastases can potentially be treated through a systemic delivery routes, such as vasculature, to allow for access to all body sites were metastatic tumors may reside. However, upon using this route to achieve systemic adenovirus delivery, over 90% of the administered vector dose is rapidly sequestered by the liver, leading to virus inactivation, reducing the efficacy of extra-hepatic gene transfer, and triggering systemic innate immune and inflammatory responses. Although the in vitro-derived model of Ad cell infection postulates key roles for Ad fiber and penton proteins in mediating virus entry into cells, our in vivo analyses demonstrate that after intravascular delivery, the major Ad capsid protein - hexon - plays the principal mechanistic role in driving virus sequestration in the liver and hepatocyte transduction. Importantly, our preliminary studies strongly suggest that specific interactions of circulating antibodies with solvent-exposed hyper-variable hexon loops mechanistically define virus interaction with Kupffer cells, leading to virus trapping in the liver and inactivation. Furthermore, our preliminary studies also demonstrated that only simultaneous inactivation of adenovirus interactions with hepatocytes, sinusoid endothelial cells, and Kupffer cells allows for virus escape from being sequestered in the liver after intravascular delivery. Although Ad vectors that are attenuated at either hepatocyte transduction or interaction with Kupffer cells have been described, to date, there are no studies published that provide direct and definitive evidence that such vectors escape liver sequestration shortly after intravascular injection. Based on the novel concept of equifunctional role of different hepatocellular compartments in sequestering Ad from the blood, in this proposal we will fill the gap in our knowledge of the role of Ad hexon in guiding virus bio- distribution an infectivity after intravascular delivery. Through a combination of structural cryo-electron- microscopy (cryo-EM) and computational methods of analysis and site-directed mutagenesis, in this proposal we will 1) determine the surface regions of adenovirus hexon that interact with low affinity natural antibodies (IgM) and high affinity mouse and human antibodies (IgG). We will also 2) determine the role of the hexon HVR1 loop variation in virus infection, replication, and Kupffer cell trapping. Finally, using a set of unique vectors with modified pentons and hexons, we will 3) develop novel hexon-mutated viruses that will avoid Kupffer cell trapping and resist neutralization with virus-specific antibodies after intravascular delivery. Our hypothesis and data-driven studies proposed in this application will greatly advance our understanding of Ad hexon - host cell and factor interactions in vivo and should ultimately lead to the experimental validation of novel strategies to prevent Ad sequestration from the blood. Conceptual and experimental validation of these strategies would represent a major step toward the development of safe and effective systemically-applicable Ad vectors for numerous therapeutic applications in humans.
The overall goals of this grant proposal are i) to develop an integrated comprehensive mechanistic model of Ad hexon interaction with neutralizing and non-neutralizing natural antibodies that leads to virus trapping in Kupffer cells and inactivation; and ii) to develop a novel class of clinically useful, safe and effective Ad vectors capable of stably circulating in the blood and amenable to extra-hepatic cell targeting following intravascular delivery. Upon targeting to tumor cells in vivo, these vectors should demonstrate increased tumor cell transduction at lower administered doses.
|Mandal, Pratyusha; Feng, Yanjun; Lyons, John D et al. (2018) Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock. Immunity 49:42-55.e6|
|Di Paolo, Nelson C; Shayakhmetov, Dmitry M (2016) Interleukin 1? and the inflammatory process. Nat Immunol 17:906-13|
|Nemerow, Glen R; Stewart, Phoebe L (2016) Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity. Viruses 8:|
|Ciraci, Ceren; Janczy, John R; Jain, Nidhi et al. (2016) Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo. PLoS One 11:e0151252|
|Atasheva, Svetlana; Shayakhmetov, Dmitry M (2016) Adenovirus sensing by the immune system. Curr Opin Virol 21:109-113|
|Di Paolo, Nelson C; Shafiani, Shahin; Day, Tracey et al. (2015) Interdependence between Interleukin-1 and Tumor Necrosis Factor Regulates TNF-Dependent Control of Mycobacterium tuberculosis Infection. Immunity 43:1125-36|
|Aiyegbo, Mohammed S; Eli, Ilyas M; Spiller, Benjamin W et al. (2014) Differential accessibility of a rotavirus VP6 epitope in trimers comprising type I, II, or III channels as revealed by binding of a human rotavirus VP6-specific antibody. J Virol 88:469-76|
|Puppo, A; Cesi, G; Marrocco, E et al. (2014) Retinal transduction profiles by high-capacity viral vectors. Gene Ther 21:855-65|