FX and Host Defense Evasion Tactics by Adenovirus
Authors: Andrew H Baker, Stuart A Nicklin, Dmitry M Shayakhmetov
Journal: Molecular Therapy (2013)

Understanding the molecular and cellular determinants that drive interactions between a host organism and a virus are fundamental questions for both virologists and gene therapists. This rationale is directed from a virology standpoint to gain vital clues into drivers of virus-induced host cell and organ infection and to define potential antiviral strategies. For therapy, such research is utilized to guide strategies for gene-based therapeutics using vectors based on the parental virus's infectivity profile. In a recent issue of Nature Medicine, Andrew Byrnes and co-workers (Xu et al.1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar) unveiled a mechanism of adenovirus-mediated evasion against host-derived natural antibodies that is driven through binding of host-derived coagulation factor X (FX) to the adenovirus capsid. This is an important finding that has potentially far-reaching implications for understanding adenovirus-induced pathogenesis and for engineering adenovirus-based therapeutics. Adenovirus-based vectors, notably those based on serotype 5 (Ad5), are very popular in laboratory-based studies and clinical gene therapy. Landmark studies in the 1990s demonstrated that the trimeric fiber protein that projects from the virus's capsid bound to the coxsackie and adenovirus receptor (CAR) and that the penton base (situated at the base of the fiber trimer) was responsible for cellular internalization, the latter through an RGD interaction with cellular integrins.2Bergelson JM Cunningham JA Droguett G Kurt-Jones EA Krithivas A Hong JS et al.Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2620) Google Scholar,3Tomko RP Xu R Philipson L HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.Proc Natl Acad Sci USA. 1997; 94: 3352-3356Crossref PubMed Scopus (1034) Google Scholar,4Wickham TJ Mathias P Cheresh DA Nemerow GR Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1953) Google Scholar This was the classic two-step binding and internalization pathway that underpinned Ad5-based gene therapy research for many years. Strategies in the late 1990s and early 2000s therefore exploited these known pathways for gene therapy. Yet, for researchers interested in directly injecting Ad5 into the bloodstream, this classic pathway began to falter. Instead, it was observed in many (but not all) studies that liver gene transfer (the major site of sequestration and gene transfer of Ad5 following this delivery route) was not affected by modifications of the virus to block either—or both—CAR and integrin interactions (reviewed in ref. 5Nicklin SA Wu E Nemerow GR Baker AH The influence of adenovirus fiber structure and function on vector development for gene therapy.Mol Ther. 2005; 12: 384-393Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Thus, the search for alternative pathways that defined liver gene transfer gained pace. The situation was further complicated by the multitude of other interactions that were documented between Ad5 and the host following injection into the bloodstream, for example, rapid Kupffer cell uptake and interactions with platelets and red blood cells (reviewed in ref. 6Coughlan L Alba R Parker AL Bradshaw AC McNeish IA Nicklin SA et al.Tropism-modification strategies for targeted gene delivery using adenoviral vectors.Viruses. 2010; 2: 2290-2355Crossref PubMed Scopus (85) Google Scholar). The first link to adenovirus interactions with host-derived factors came from Shayakhmetov and co-workers,7Shayakhmetov DM Gaggar A Ni SH Li ZY Lieber A Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity.J Virol. 2005; 79: 7478-7491Crossref PubMed Scopus (355) Google Scholar who defined an interaction between coagulation factor IX, as well as other host-derived serum proteins and the adenovirus capsid, thereby proposing the hypothesis that additional interactions "bridged" the virus to alternative cellular receptors present on the surface of hepatocytes. An intense period in adenovirus research came to fruition in 2008 when three groups independently reported8Kalyuzhniy O Di Paolo NC Silvestry M Hofherr SE Barry MA Stewart PL Shayakhmetov DM Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo.Proc Natl Acad Sci USA. 2008; 105: 5483-5488Crossref PubMed Scopus (284) Google Scholar,9Waddington SN Mcvey JH Bhella D Parker AL Barker K Atoda H et al.Adenovirus serotype 5 hexon mediates liver gene transfer.Cell. 2008; 132: 397-409Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar,10Vigant F Descamps D Jullienne B Esselin S Connault E Opolon P et al.Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver.Mol Ther. 2008; 16: 1474-1480Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar that an interaction of FX with the adenovirus's trimeric hexon protein—the major adenovirus capsid protein—was responsible for bridging to hepatic receptors (subsequently confirmed as heparan sulfate proteoglycans11Bradshaw AC Parker AL Duffy MR Coughlan L van Rooijen N Kähäri V-M et al.Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.PLoS Pathog. 2010; 6: e1001142Crossref PubMed Scopus (66) Google Scholar). Indeed, mutations that selectively blocked this high-affinity interaction ablated liver gene transfer.12Alba R Bradshaw AC Coughlan L Denby L McDonald RA Waddington SN et al.Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.Blood. 2010; 116: 2656-2664Crossref PubMed Scopus (95) Google Scholar,13Alba R Bradshaw AC Parker AL Bhella D Waddington SN Nicklin SA et al.Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer.Blood. 2009; 114: 965-971Crossref PubMed Scopus (151) Google Scholar,14Doronin K Flatt JW Di Paolo NC Khare R Kalyuzhniy O Acchione M et al.Coagulation factor X activates innate immunity to human species C adenovirus.Science. 2012; 338: 795-798Crossref PubMed Scopus (126) Google Scholar Notably, eliminating this interaction did not alter the interaction of adenovirus with Kupffer cells in the liver, an antiviral scavenging pathway.15Waddington SN Parker AL Havenga M Nicklin SA Buckley SMK Mcvey JH et al.Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5.J Virol. 2007; 81: 9568-9571Crossref PubMed Scopus (68) Google Scholar There is therefore absolutely no doubt that Ad5 binding to FX mediates liver gene transfer in a fully immune-competent setting, a finding observed in at least three species.9Waddington SN Mcvey JH Bhella D Parker AL Barker K Atoda H et al.Adenovirus serotype 5 hexon mediates liver gene transfer.Cell. 2008; 132: 397-409Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar,16Alba R Bradshaw AC Mestre-Frances N Verdier JM Henaff D Baker AH Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus).Gene Ther. 2012; 19: 109-113Crossref PubMed Scopus (38) Google Scholar Recent studies have shown that this Ad5–FX interaction also activates immune regulation in the host.14Doronin K Flatt JW Di Paolo NC Khare R Kalyuzhniy O Acchione M et al.Coagulation factor X activates innate immunity to human species C adenovirus.Science. 2012; 338: 795-798Crossref PubMed Scopus (126) Google Scholar Furthermore, it has been confirmed that some, but not all, other human adenoviruses bind to coagulation factors,9Waddington SN Mcvey JH Bhella D Parker AL Barker K Atoda H et al.Adenovirus serotype 5 hexon mediates liver gene transfer.Cell. 2008; 132: 397-409Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar thus suggesting an interaction that has potentially evolved in evolutionary terms. Although this interaction was observed in the context of bolus adenovirus injection in the bloodstream (i.e., in the setting of adenovirus-mediated gene therapy), the key question is why adenovirus binds to FX in the first place. Is this a host defense mechanism against adenovirus dissemination through the bloodstream, a key event in pathogenic adenovirus spread? Or is this a strategy utilized by the virus to evade host interactions that might disable or limit adenovirus dissemination? The paper by Xu et al.1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar suggests the latter. They describe a set of remarkable observations that drastically add to the complexity of adenovirus interaction with the host after intravascular delivery. In contrast to the aforementioned FX bridging mechanism, the authors report that Ad5 devoid of the capacity to bind to FX enters hepatocytes efficiently in mice that lack natural IgM antibodies or complement components C1q and C4 (but not in normal mice). Through a set of elegant experiments, the authors showed that complement component C3b is deposited onto the virus surface in an IgM-, C1q-, and C4-dependent manner, and that this deposition reduces the ability of the virus to attach to susceptible cells in vitro. The nonmodified Ad5, when in complex with FX, failed to interact with complement C3b. The authors therefore concluded that FX has another, previously unknown function as an inhibitor of Ad5-induced complement activation and that FX binding is an effective strategy utilized by Ad5 to avoid being attacked by natural antibodies and complement. These findings are of great importance, and the ramifications are highly significant for both our understanding of adenovirus–host interactions and the development of novel approaches for efficient cell type–specific targeting of adenovirus vectors using an intravascular route of delivery. Two fundamental questions arise from the findings. First, what is the mechanism of Ad5 entry into hepatocytes in vivo in the setting of animals that lack IgM or complement components? Second, does IgM-mediated deposition of complement on FX-deficient Ad5 vectors lead to virus inactivation, thereby rendering it potentially useless as a vector for delivering therapeutic genes to extrahepatic sites? The definitive answers to these questions are plagued by the inconsistencies between cell responses to adenovirus observed in in vitro and in vivo systems. Whereas in vitro CAR is critical for efficient virus entry into the cell,2Bergelson JM Cunningham JA Droguett G Kurt-Jones EA Krithivas A Hong JS et al.Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2620) Google Scholar,3Tomko RP Xu R Philipson L HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.Proc Natl Acad Sci USA. 1997; 94: 3352-3356Crossref PubMed Scopus (1034) Google Scholar it is dispensable for virus entry into at least some cell types, including hepatocytes, in vivo.7Shayakhmetov DM Gaggar A Ni SH Li ZY Lieber A Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity.J Virol. 2005; 79: 7478-7491Crossref PubMed Scopus (355) Google Scholar Ad5 can activate complement through natural antibodies via the classic pathway in vitro,17Appledorn DM McBride A Seregin S Scott JM Schuldt N Kiang A et al.Complex interactions with several arms of the complement system dictate innate and humoral immunity to adenoviral vectors.Gene Ther. 2008; 15: 1606-1617Crossref PubMed Scopus (63) Google Scholar but complement activation in vivo depends on the reticuloendothelial system and occurs via both the classical and alternative pathways independently of natural antibodies.18Tian J Xu ZL Smith JS Hofherr SE Barry MA Byrnes AP Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo.J Virol. 2009; 83: 5648-5658Crossref PubMed Scopus (72) Google Scholar Although macrophages in the liver and spleen efficiently scavenge adenovirus19Shayakhmetov DM Li ZY Ni S Lieber A Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors.J Virol. 2004; 78: 5368-5381Crossref PubMed Scopus (177) Google Scholar,20Di Paolo NC Miao EA Iwakura Y Murali-Krishna K Aderem A Flavell RA et al.Virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo.Immunity. 2009; 31: 110-121Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar and those cells rapidly die in vivo,21Manickan E Smith JS Tian J Eggerman TL Lozier JN Muller J et al.Rapid Kupffer cell death after intravenous injection of adenovirus vectors.Mol Ther. 2006; 13: 108-117Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar macrophages in vitro are resistant to virus infection and do not die even after exposure to very high amounts of the virus.22Nociari M Ocheretina O Murphy M Falck-Pedersen E Adenovirus induction of IRF3 occurs through a binary trigger targeting Jun N-terminal kinase and TBK1 kinase cascades and type I interferon autocrine signaling.J Virol. 2009; 83: 4081-4091Crossref PubMed Scopus (35) Google Scholar Therefore, in our attempt to answer the two fundamental questions above in light of the data described by Xu et al.,1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar we focus this Commentary entirely on the data obtained from the in vivo systems utilized in the study. First, the data presented by the authors do not exclude the principal role of FX in mediating Ad5 entry into hepatocytes in vivo in normal, immune-competent mice. However, whether FX can indeed function as the one and only molecule that bridges the virus to hepatocellular receptors in the in vivo system remains unclear, even though FX clearly plays a predominant role in leading to effective hepatic gene transfer. One previous study showed that in vivo liver transduction by Ad5-based vectors is greatly reduced in mice pretreated with heparinase or lactoferrin,7Shayakhmetov DM Gaggar A Ni SH Li ZY Lieber A Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity.J Virol. 2005; 79: 7478-7491Crossref PubMed Scopus (355) Google Scholar suggesting that specific receptors that mediate uptake of adenovirus particles from the blood can be inhibited by both of these treatments. The definitive answer may come from analyzing hepatocyte transduction by nonmodified Ad5 in complex with FX with a mutated HSPG-binding exosite, as described by Duffy et al.,23Duffy MR Bradshaw AC Parker AL Mcvey JH Baker AH A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.J Virol. 2011; 85: 10914-10919Crossref PubMed Scopus (31) Google Scholar but in vivo in warfarin-treated mice. Second, the answer to the question as to whether IgM-mediated deposition of complement truly reduces virus infectivity and/or bioavailability in vivo is more definitive. Although extensive in vitro analyses by Xu et al.1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar strongly argue that IgM-mediated complement fixation reduces virus capacity to bind to susceptible cells, other groups reported that in vivo, when FX binding to Ad is blocked via warfarin pretreatment of mice or hexon mutations, the virus becomes de-targeted from hepatocytes and transduces other cell populations, including endothelial cells and immune cells in different organs.12Alba R Bradshaw AC Coughlan L Denby L McDonald RA Waddington SN et al.Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.Blood. 2010; 116: 2656-2664Crossref PubMed Scopus (95) Google Scholar,24Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-borne adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar,25Bradshaw AC Coughlan L Miller AM Alba R van Rooijen N Nicklin SA et al.Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses.J Control Release. 2012; 164: 394-402Crossref PubMed Scopus (35) Google Scholar Furthermore, the most definitive data were obtained on analyzing transduction of human tumor xenografts in mice by FX-binding and FX binding–ablated oncolytic adenoviruses, or in tumor-bearing mice after warfarin treatment.26Shashkova EV Doronin K Senac JS Barry MA Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus.Cancer Res. 2008; 68: 5896-5904Crossref PubMed Scopus (81) Google Scholar,27Shashkova EV May SM Doronin K Barry MA Expanded anticancer therapeutic window of hexon-modified oncolytic adenovirus.Mol Ther. 2009; 17: 2121-2130Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Of note, in immunodeficient nude mice, in which Xu et al. showed that Ad inhibitory activity in the blood is as potent as in control C57BL6 mice,1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar the accumulation of virus in tumor xenografts and the antitumor activity of oncolytic Ad were significantly higher following pretreatment of mice with warfarin,26Shashkova EV Doronin K Senac JS Barry MA Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus.Cancer Res. 2008; 68: 5896-5904Crossref PubMed Scopus (81) Google Scholar which inactivates all vitamin K–dependent circulating coagulation factors, including FX. Collectively, these studies show improved oncolytic efficacy of the viruses under the conditions when FX binding was inactivated, providing direct evidence that, even without binding of coagulation factors, Ad vectors retain at least some level of infectivity after intravascular delivery. Whether further improved uptake via direct targeting strategies may be limited by virus inactivation remains to be robustly tested in the in vivo setting. The study by Xu et al. has undoubtedly revealed a new layer of complexity of Ad interaction with the host after intravascular virus administration. It is important to consider that within minutes after intravascular administration, FX binding–ablated viruses are trapped in the liver tissue in amounts identical to those of FX-binding wild-type vectors.12Alba R Bradshaw AC Coughlan L Denby L McDonald RA Waddington SN et al.Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.Blood. 2010; 116: 2656-2664Crossref PubMed Scopus (95) Google Scholar,15Waddington SN Parker AL Havenga M Nicklin SA Buckley SMK Mcvey JH et al.Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5.J Virol. 2007; 81: 9568-9571Crossref PubMed Scopus (68) Google Scholar,24Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-borne adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar This indicates that even though Ad entry into hepatocytes is inefficient owing to lack of FX or its inactivation with warfarin treatment, virus particles—now suggested to be in complex with IgM, C1q, and C4—are still retained primarily in the liver tissue, albeit probably in a different cellular compartment. Therefore, to unify all the available data into a single model that would explain Ad infectivity and biodistribution after intravascular delivery, we propose that when Ad5 is injected intravenously and is able to bind FX, it is first delivered to liver Kupffer cells and inactivated. Following Kupffer cell saturation, Ad5–FX complexes are targeted to hepatocytes via a "bridge" mechanism that results in highly efficient FX-mediated hepatocyte transduction through an FX-mediated interaction with HSPGs11Bradshaw AC Parker AL Duffy MR Coughlan L van Rooijen N Kähäri V-M et al.Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.PLoS Pathog. 2010; 6: e1001142Crossref PubMed Scopus (66) Google Scholar (Figure 1). However, when FX binding is ablated via genetic mutations of the hexon13Alba R Bradshaw AC Parker AL Bhella D Waddington SN Nicklin SA et al.Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer.Blood. 2009; 114: 965-971Crossref PubMed Scopus (151) Google Scholar,14Doronin K Flatt JW Di Paolo NC Khare R Kalyuzhniy O Acchione M et al.Coagulation factor X activates innate immunity to human species C adenovirus.Science. 2012; 338: 795-798Crossref PubMed Scopus (126) Google Scholar or warfarin pretreatment of the host, Ad5 is coated with IgM and complement components C1q, C4, and C3. These new immunocomplexes also target Ad to Kupffer cells,28Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar and potentially other cells in the liver or the circulation that express complement receptors, and preclude any liver-directed gene transfer because FX is not available. After intravascular delivery of Ad that cannot interact with FX into mice deficient in IgM or complement, it is not targeted to complement receptor-expressing cells but instead remains in the circulation and may enter hepatocytes via macropinocytosis or other, as-yet unknown mechanisms, leading to efficient hepatocyte transduction. Ad trapping by circulating and/or liver-localized neutrophils in a complement receptor–dependent manner has previously been shown.29Cotter MJ Zaiss AK Muruve DA Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1.J Virol. 2005; 79: 14622-14631Crossref PubMed Scopus (60) Google Scholar Furthermore, it was recently found that natural IgM mediates complement-dependent uptake (but not inactivation) of bacterial pathogen Francisella tularensis by human neutrophils,30Schwartz JT Barker JH Long ME Kaufman J McCracken J Allen LAH Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via complement receptors 1 and 3 in nonimmune serum.J Immunol. 2012; 189: 3064-3077Crossref PubMed Scopus (42) Google Scholar suggesting that IgM complement–mediated opsonization may represent a universally utilized default mechanism for pathogen removal from the blood. Collectively, the findings by Xu et al.1Xu Z Qiu Q Tian J Smith JS Conenello GM Morita T et al.Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.Nat Med. 2013; 19: 452-457Crossref PubMed Scopus (127) Google Scholar vividly demonstrate that Ad interaction with natural antibodies and complement is another significant barrier to contemplate when designing Ad-based vectors for gene therapy via the intravascular route. Important questions remain to be answered in this complex but important area of Ad5 biology. Are other Ad species that do not naturally bind FX (or bind at lower affinities than Ad5) inactivated by complement in the bloodstream, or are there additional mechanisms of protection following interaction with other host factors? How all these factors interplay in the human system remains unclear, because disseminating Ad infections are the major complication in patients with compromised immune function. It is clear that future research will add to this important and topical area of Ad biology, with important implications for both Ad-mediated gene therapy and basic knowledge regarding Ad–host interaction.