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De-livering an oncolytic virus: Oncolytic viruses, optimized for killing tumor cells, are a promising modality in cancer immunotherapy. However, there are some challenges that these viruses must overcome, including antiviral immune responses that can affect both the efficacy and the safety of these viruses. For example, naturally occurring immunoglobulin M (IgM) antibodies bind to human species C adenovirus, resulting in trapping of the virus in the liver. To overcome this, Atasheva et al. engineered a modified version of this adenovirus, with mutations in the binding site for IgM. This modified virus was not trapped in the liver and effectively suppressed tumor growth in mouse models without safety concerns. Oncolytic virus therapy is a cancer treatment modality that has the potential to improve outcomes for patients with currently incurable malignancies. Although intravascular delivery of therapeutic viruses provides access to disseminated tumors, this delivery route exposes the virus to opsonizing and inactivating factors in the blood, which limit the effective therapeutic virus dose and contribute to activation of systemic toxicities. When human species C adenovirus HAdv-C5 is delivered intravenously, natural immunoglobulin M (IgM) antibodies and coagulation factor X rapidly opsonize HAdv-C5, leading to virus sequestration in tissue macrophages and promoting infection of liver cells, triggering hepatotoxicity. Here, we showed that natural IgM antibody binds to the hypervariable region 1 (HVR1) of the main HAdv-C5 capsid protein hexon. Using compound targeted mutagenesis of hexon HVR1 loop and other functional sites that mediate virus-host interactions, we engineered and obtained a high-resolution cryo–electron microscopy structure of an adenovirus vector, Ad5-3M, which resisted inactivation by blood factors, avoided sequestration in liver macrophages, and failed to trigger hepatotoxicity after intravenous delivery. Systemic delivery of Ad5-3M to mice with localized or disseminated lung cancer led to viral replication in tumor cells, suppression of tumor growth, and prolonged survival. Thus, compound targeted mutagenesis of functional sites in the virus capsid represents a generalizable approach to tailor virus interactions with the humoral and cellular arms of the immune system, enabling generation of "designer" viruses with improved therapeutic properties. [ABSTRACT FROM AUTHOR]

Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application. Major obstacles to clinical use include innate toxicity and Ad sequestration by nontarget tissues. The factors and mechanisms underlying these processes are poorly understood. The majority of intravenously injected Ad particles are sequestered by the liver, which in turn causes an inflammatory response characterized by acute transaminitis and vascular damage. Here, we describe a novel pathway that is used by Ad for infection of hepatocytes and Kupffer cells upon intravenous virus application in mice. We found that blood factors play a major role in targeting Ad vectors to hepatic cells. We demonstrated that coagulation factor IX and complement component C4-binding protein can bind the Ad fiber knob domain and provide a bridge for virus uptake through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. An Ad vector, Ad5mut, which contained mutations in the fiber knob domain ablating blood factor binding, demonstrated significantly reduced infection of liver cells and liver toxicity in vivo. This study contributes to a better understanding of adenovirus-host interactions for intravenously applied vectors. It also provides a rationale for novel strategies to target adenovirus vector to specific tissues and to reduce virus-associated toxicity after systemic application. [ABSTRACT FROM AUTHOR]

Human adenovirus (Ad) is a ubiquitous pathogen causing a wide range of diseases. Although the interactions of human Ad serotype 5 (Ad5) with susceptible cells in vitro are known in great detail, host factors controlling the tissue specificity of Ad5 infection in vivo remain poorly understood. Here, we analyzed the mechanisms of sequestration by the liver for blood-born human Ads and Ad5-based vectors. Our data suggest that several known mechanisms that lead to Ad5 sequestration by the liver become engaged in a redundant, sequential, and synergistic manner to ensure the rapid clearance of circulating virus particles from the blood. These mechanisms include (i) trapping of the virus by liver residential macrophages, Kupffer cells; (ii) Ad5 hepatocyte infection via blood factor–hexon interactions; and (iii) Ad5 penton RGD motif–mediated interactions with liver endothelial cells and hepatocytes, mediating virus retention in the space of Disse. More important, we show that when all of these mechanisms are simultaneously inactivated via mutations of Ad5 capsid proteins and pharmacological interventions, virus sequestration by the liver is markedly reduced. Therefore, our study is the first demonstration of the principal possibility of ablating the sequestration of blood-born Ad in the liver via specific inactivation of a defined set of mechanisms that control this process.Molecular Therapy (2009) 17 4, 675–684 doi:10.1038/mt.2008.307 [ABSTRACT FROM AUTHOR]

The article discusses research being done on adenovirus-mediated evasion against host-derived natural antibodies. It references the study "Coagulation Factor X Shields Adenovirus Type 5 From Attack by Natural Antibodies and Complement," by Z. Xu et al. in the 2013 issue of "Nature Medicine." The researcher observed that host-derived coagulation factor X (FX) has linked to the adenovirus capsid and entered hepatocytes in mice without natural immunoglobulin M (IgM) antibodies.

Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1HEP mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1HEP mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1HEP mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. [ABSTRACT FROM AUTHOR]

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus. [ABSTRACT FROM AUTHOR]

The molecular mechanisms governing the infectivity of adenovirus (Ad) toward specific cell and tissue types in vivo remain poorly understood. The direct Ad binding to hepatic heparan sulfate proteoglycans via the KKTK motif within the fiber shaft domain was suggested to be the major mechanism of Ad liver cell infection in vivo. Here, we describe the generation and in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains, which lack the KKTK motif. We found that all the critical early steps of Ad infection, including attachment to the cellular receptor, internalization, and virus genome transfer into the nucleus, occurred with similar levels of efficiency for fiber shaft-chimeric vectors and unmodified Ad5. Upon intravenous delivery into mice, fiber shaft-chimeric vectors accumulated in liver tissue, transduced liver cells, and induced the production of proinflammatory cytokines (tumor necrosis factor alpha and interleukin-6) and the chemokine monocyte chemoattractant protein 1 at levels indistinguishable from those observed for Ad5. Thus, our data provide evidence that the Ad5 fiber shaft amino acid sequence does not play any substantial role in determining adenovirus infectivity toward hepatic cells in vivo. The data obtained contribute to improving our understanding of the molecular mechanisms determining Ad infectivity and biodistribution in vivo and may aid in designing novel Ad-based vectors for gene therapy applications. [ABSTRACT FROM AUTHOR]

Intensive studies of adenovirus infectivity and bio-distribution in animal models demonstrated that liver tissue is responsible for clearance of the bulk of intravenously applied vector. We recently demonstrated that adenovirus binding to blood factors, specifically coagulation factor IX and C4 binding protein, leads to high level hepatocyte transduction and virus trapping by Kupffer cells. Apparently, virus interaction with the coxsackie- and adenovirus receptor, CAR, was not required for virus trapping by hepatic cells, although the fiber knob domain was identified as the primary moiety, mediating virus interaction with blood factors in vivo. Because adenovirus hepatocyte transduction and trapping by Kupffer cells is associated with the severe innate immune and inflammatory host responses observed following systemic adenovirus administration, we hypothesized that preventing virus interaction with blood factors should dramatically improve both the circulatory persistence and toxicity profile of adenovirus vectors. To better understand the structural basis of Ad fiber knob domain interaction with FIX, we first analyzed the efficiency of FIX-Ad knob association at different ionic strengths using dynamic light scattering. These analyses revealed that formation of the Ad fiber knob-FIX complex is highly sensitive to ionic strength and most efficient at low salt concentrations. Salt concentrations exceeding 0.5M, on the other hand, completely prevent complex formation. These data suggest that interaction between hydrophilic charged amino acids in FIX and Ad fiber knob domain, rather than hydrophobic amino acids, plays the major role in complex formation. To further identify the precise areas of protein-protein interaction, we cross-linked FIX with purified recombinant Ad5 or Ad9 fiber knob domains. Using PAGE protein gels we confirmed that upon incubation with different cross linkers, FIX and Ad fiber knobs efficiently form complexes that migrate within the expected mass range. Proteolytic digestion of cross-linked Ad fiber knob-FIX complex and subsequent analysis of obtained peptides using ESI-MS and MS/MS revealed several candidate peptides participating in the cross-linking reaction. These peptides define the interface of Ad fiber knob-FIX interaction. To confirm mass spectrometry analysis data, we are currently generating a panel of Ad fiber knobs possessing amino acid mutations within the putative protein interface area. The approach of protein cross-linking followed by mass spectrometry analyses may be widely applied to study interactions of virus proteins with host factors in vitro and in vivo. The development of Ad vectors ablated for binding to blood factors should ultimately allow for the construction of safe and efficient adenovirus vectors for therapeutic application in humans.Molecular Therapy (2006) 13, S144–S144; doi: 10.1016/j.ymthe.2006.08.439 [ABSTRACT FROM AUTHOR]