Binding of adenovirus species C hexon to prothrombin and the influence of hexon on vector properties in vitro and in vivo.

The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.

Interaction of adenovirus with antibodies, complement, and coagulation factors.

Adenovirus (AdV) is one of the most widely used vectors for gene therapy and vaccine studies due to its excellent transduction efficiency, capacity for large transgenes, and high levels of gene expression. When administered intravascularly, the fate of AdV vectors is heavily influenced by interactions with host plasma proteins. Some plasma proteins can neutralize AdV, but AdV can also specifically bind plasma proteins that protect against neutralization and preserve activity. This review summarizes the plasma proteins that interact with AdV, including antibodies, complement, and vitamin K-dependent coagulation factors. We will also review the complex interactions of these plasma proteins with each other and with cellular proteins, as well as strategies for developing better AdV vectors that evade or manipulate plasma proteins.

Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum.

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors.

Evaluating the Impact of Natural IgM on Adenovirus Type 5 Gene Therapy Vectors.

Natural IgM antibodies have an innate ability to recognize many viruses and viral-based gene therapy vectors. Naive mice have natural IgM antibodies that bind to adenoviruses, and these antibodies can profoundly affect the biodistribution and efficiency of gene delivery by adenovirus type 5 vectors. Here, we present protocols for isolating IgM from mouse serum, for assaying the concentration and adenoviral reactivity of mouse IgM, and for evaluating how natural antibodies and complement can synergize to neutralize adenovirus vectors.

Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors.

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.

Adenovirus type 5 (Ad5) specifically binds coagulation factor X (FX), and FX is normally essential for intravenously injected Ad5 vectors to transduce the liver. We demonstrate that the ability of FX to enhance liver transduction by Ad5 vectors is due to an unexpected ability of FX to protect Ad5 from attack by the classical complement pathway. In vitro, naive mouse serum neutralized Ad5 when FX was blocked from binding Ad5. This neutralization was mediated by natural IgM and the classical complement pathway. In vivo, FX was essential for Ad5 vectors to transduce the livers of wild-type mice, but FX was not required for liver transduction in mice that lack antibodies, C1q or C4. We conclude that Ad5 recruits FX as a defense against complement and that the sensitivity of Ad5 to inactivation by complement must be taken into account when designing vectors for systemic gene therapy.

Circulating antibodies and macrophages as modulators of adenovirus pharmacology.

Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.

The role of endosomal escape and mitogen-activated protein kinases in adenoviral activation of the innate immune response.

Adenoviral vectors (AdV) activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs). In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK) and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases.

Breakout session C summary: current virus detection methods.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA).

Induction of shock after intravenous injection of adenovirus vectors: a critical role for platelet-activating factor.

Innate immune responses are a major barrier to safe systemic gene therapy with adenovirus (Ad) vectors. We show that intravenous (IV) injection of rats with Ad5 vectors causes a novel rapid shock reaction that involves hypotension, hemoconcentration, tissue edema, and vasocongestion, with notable pathology in the pancreas and the gastrointestinal system. We show for the first time that this reaction is dependent on platelet-activating factor (PAF), a lipid signaling molecule that is a known shock inducer. Ad upregulated PAF within 5 minutes in vivo, and antagonists of the PAF receptor were able to prevent Ad-induced shock. Ad upregulated PAF via the reticuloendothelial system (RES), because splenectomy or depletion of phagocytes blocked the ability of Ad to induce both PAF and shock. Rats were considerably more sensitive to Ad-induced shock than were mice, but PAF mediated shock in both species. Other Ad-induced innate immune responses such as cytokine induction and thrombocytopenia were not mediated by PAF. In summary, systemic IV injection of Ad stimulates the RES to upregulate PAF within a matter of minutes, which results in shock. The identification of this novel pathway suggests strategies to improve the safety of systemic gene therapy with Ad vectors.

Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo.

Understanding innate immunity is key to improving the safety of adenovirus (Ad) vectors for systemic gene therapy. Ad has been shown to activate complement in vitro, but activation of complement after Ad injection in vivo has not been directly measured. Using complement protein C3a as a marker of complement activation, we show that types 2 and 5 human Ads cause rapid complement activation after intravenous injection in mice. Unexpectedly, the mechanisms in vivo were different than those in vitro. Antibodies were critical for the activation of complement by Ad in vitro, but antibodies were not required in vivo. The classical pathway was required in vitro, whereas complement activation in vivo involved both classical and nonclassical pathways as well as the reticuloendothelial system. Remarkably, the entry-deficient Ad mutant ts1 was completely unable to activate complement in vivo even though it was fully able to activate complement in vitro. This result demonstrates that the complement system senses intravenously injected Ad primarily by detecting the effects of Ad on cells rather than through direct interaction of complement with virions. Encouragingly, shielding Ad with polyethylene glycol was effective at reducing complement activation both in vitro and in vivo. In summary, intravenously injected Ad rapidly activates complement through multiple pathways, but these pathways are different than those identified by in vitro studies. In vitro studies are poorly predictive of in vivo mechanisms because Ad virions activate complement through indirect mechanisms in vivo.

Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.

Kupffer cells (KCs) rapidly remove intravenously injected adenovirus (Ad) vectors from the circulation. A better understanding of the mechanisms involved could suggest strategies to improve Ad gene delivery by suppressing or evading KC uptake. We recently showed that clearance of Ad type 5 vectors by KCs does not involve the interaction of Ad with the well-established Ad receptors, namely, integrins or the coxsackievirus and Ad receptor (J. S. Smith, Z. Xu, J. Tian, S. C. Stevenson, and A. P. Byrnes, Hum. Gene Ther. 19:547-554, 2008). In the current study, we systematically quantified the contributions of various receptors and plasma proteins to the clearance of Ad by KCs. We found that scavenger receptors are a predominant mechanism for the clearance of Ad by KCs. In addition, we found that Ad is opsonized by natural immunoglobulin M antibodies and complement and that these opsonins play a contributory role in the clearance of Ad by KCs. We also examined additional mechanisms that have been postulated to be involved in the clearance of Ad, including the binding of Ad to platelets and vitamin K-dependent coagulation factors, but we found that neither of these were required for the clearance of Ad by KCs.

Th1 cytokines are upregulated by adenoviral vectors in the brains of primed mice.

A major disadvantage of first generation adenoviral vectors for gene therapy in the brain is the immune response they elicit. Human adenovirus is a common respiratory virus and earlier exposure to it has important implications for gene therapy. We show that the immune response against E1-deleted adenoviral vectors in the brain is more deleterious in animals previously exposed to the virus. Analysis of cytokine mRNA revealed enhanced and prolonged upregulation of the Th1 proinflammatory cytokines, IFN-gamma, TNF-alpha and IL-12 whereas, effects on Th2 cytokines were negligible. This was associated with reduced reporter gene expression, decreased expression of the dopamine transporter protein and demyelination. This knowledge of the molecular regulation of the immune response provides insight into targets, which could be manipulated to reduce inflammation in immunologically primed animals.

Interaction of systemically delivered adenovirus vectors with Kupffer cells in mouse liver.

When adenovirus (Ad) vectors are injected intravenously they are rapidly taken up by Kupffer cells (KCs) in the liver. This results in massive KC necrosis within minutes, followed by a more gradual disappearance of KCs from the liver. It is not known how KCs recognize Ad, or why Ad kills KCs. We used a variety of mutated and fiber-pseudotyped Ad vectors to evaluate how capsid proteins influence Ad uptake by KCs and to define the viral proteins that are involved in the destruction of KCs. We found that depletion of KCs from the liver was partially dependent on interactions between Ad and integrins, but was independent of the coxsackievirus and Ad receptor. The Ad5 fiber shaft was proven to be a particularly important contributory factor, because vectors with the shorter Ad35 shaft were not as effective at depleting KCs. In contrast, the fiber head played no discernible role. Variations in the ability of Ad vectors to deplete KCs could not be explained by differences in the amount of Ad that reached KCs, because all mutant Ads were accumulated by KCs at similar levels. Interestingly, we found that the Ad mutant ts1 did not cause KC death; this virus is known to bind and enter cells normally, but the capsid is unable to disassemble or lyse membranes. We conclude that Ad vectors kill KCs at a postbinding step and that this cell death can be mitigated if downstream events in viral entry are blocked.

A quantitative assay for measuring clearance of adenovirus vectors by Kupffer cells.

Kupffer cells are a major barrier to systemic adenovirus (Ad) gene therapy because they rapidly and efficiently clear virions from the circulation. The lack of a straightforward quantitative technique for selectively measuring uptake of Ad by Kupffer cells has made it difficult to study the mechanisms by which they recognize Ad. A new method was developed that relies on immunofluorescent detection of Ad within Kupffer cells in mouse liver sections, followed by confocal microscopy and computerized image analysis. The method is sensitive, quantitative and reproducible, with a linear range spanning two orders of magnitude. As an example of the utility of this method, it was found that pre-injecting mice with polyinosinic acid reduces accumulation of Ad in Kupffer cells by approximately 90%.

Quality prediction of cell substrate using gene expression profiling.

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.

Rapid Kupffer cell death after intravenous injection of adenovirus vectors.

When adenovirus vectors are injected intravenously, they are quickly taken up by Kupffer cells in the liver. We report that this causes rapid necrosis of Kupffer cells in mice at doses of 10(11) particles/kg or higher. By 10 min after intravenous vector injection, Kupffer cells were permeable to propidium iodide and trypan blue. This coincided with a sharp rise in serum lactate dehydrogenase. Ultrastructural examination showed degeneration of Kupffer cells, including complete disappearance of chromatin by 1 h. After an initial intravenous injection of vector, dead Kupffer cells were unable to take up a second dose of vector, and hepatic transgene expression from the second dose was augmented. Death of Kupffer cells did not affect serum levels of IL-6 or IL-12. There was no immediate change in the number of Kupffer cells in the liver, but a significant decline was found by 4 h after injection of vector. Interestingly, substantial numbers of vector-containing Kupffer cells were found in pulmonary capillaries, indicating that they had been swept out of the liver. Together these results show that an intravenous injection of adenovirus vector causes synchronous and surprisingly rapid Kupffer cell death.

Heparin-binding and patterns of virulence for two recombinant strains of Sindbis virus.

E2 is an important determinant of Sindbis virus neurovirulence. Increased heparan sulfate (HS) binding is associated with rapid clearance of viremia and usually with decreased virulence. However, substitution of histidine for arginine at E2-157 (R157H) or glutamate for lysine at E2-159 (K159E) produces viruses with decreases in heparin-Sepharose binding and increases in viremia but different levels of binding to HS-expressing cells and virulence phenotypes in newborn CD-1 mice (Byrnes, A.P., Griffin, D.E., 2000. Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia and slower clearance from the circulation. J. Virol. 74, 644-651). To identify mechanisms of virulence, R157H and K159E were studied in newborn CD-1 and BALB/c mice. Subcutaneous inoculation of R157H caused 100% and K159E 60% mortality in 2-day-old CD-1 mice. R157H caused 25% and K159E no mortality in 2-day-old BALB/c mice. R157H and K159E replicated similarly at the site of inoculation with the same level of viremia, but clearance was slower in CD-1 than BALB/c mice. R157H replicated better than K159E in the central nervous system (CNS) after subcutaneous and intracerebral inoculation and in undifferentiated neurons. These studies show a genetic restriction of replication in newborn BALB/c mice, and that amino acid substitutions affecting binding to proteoglycans may differ in importance for CNS infection and viremia.

Challenges and future prospects in gene therapy.

Gene therapy is a promising technique for treating disease through the modification of gene expression. It is currently being tested not only for correcting genetic defects, but also for treating cancer and other acquired diseases. Although this field is still relatively young, evidence for clinical efficacy has been observed and continued progress seems assured, as clinical trials continue to yield insights into how gene therapy can be applied and improvements are made in gene therapy tools.

Severe pulmonary pathology after intravenous administration of vectors in cirrhotic rats.

After an intravascular injection, adenoviral vectors are normally taken up by the reticuloendothelial system in the liver, where they rapidly trigger an innate response. However, we have previously found that the biodistribution of adenoviral vectors is altered in cirrhotic rats due to the presence of pulmonary intravascular macrophages, which cause a shift in vector uptake from the liver to the lungs. We now report that this is correlated with fatal pulmonary hemorrhagic edema in cirrhotic rats. In addition, cirrhotic rats reacted to vector with enormous increases in TNF-alpha and IL-6 and markedly prolonged coagulation times. Although we also saw fatal reactions to high doses of adenoviral vectors in normal rats, the time course and symptoms were very different, and pulmonary hemorrhagic edema was seen only in cirrhotic rats. Because abnormal pulmonary reticuloendothelial uptake is known to occur in humans during cirrhosis and other diseases, there is the potential that intravascular administration of adenoviral vectors might cause lung pathology in such patients.