Adenovirus Immunity: X Marks the Spot
Authors: Masataka Suzuki, Cliona M Rooney
Journal: Molecular Therapy (2012)

Human adenovirus (HAd)-based vectors are notorious for their ability to induce profound innate immune responses, causing liver damage and even lethal circulatory collapse. In a recent issue of Science, Doronin et al.1Doronin 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 now show that one of the ways by which adenoviruses trigger the innate immune response is through the binding of coagulation factor X (FX), a phylogenetically ancient component of the blood clotting system. Interference with this process may therefore make adenoviral vectors both safer and more effective. Type C adenoviruses, such as the widely used Ad5-derived vector, have long been known to bind to FX, which forms a complex with the hexon protein in the adenoviral capsid to bind to heparan sulfate–containing glycosaminoglycans on hepatocytes and liver macrophages. However, the implications of this interaction for the triggering of innate immunity had not previously been considered. Given that FX-like molecules are important constituents of the innate immune response in many ancient phyla, Doronin et al.1Doronin 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 disrupted FX binding by mutation of the Ad5 hexon and looked for the consequences for infectivity and the innate immune response. They found that the mutant Ad5 was unable to infect hepatocytes and also failed to activate Toll-like receptor 4 (TLR4) and downstream genes in macrophages, resulting in reduced production of inflammatory cytokines and providing an evolutionary link between the coagulation system and innate immunity in higher organisms. The induction of innate immunity by HAds has been a major barrier to their use as vectors, particularly in gene correction studies. Quite apart from the acute toxicities produced by cytokine release and hepatic inflammation, the innate immune response swiftly recruits adaptive immunity, so that T cell–mediated immunity to virion proteins subsequently destroys transduced cells and high titers of neutralizing antibodies prevent effective readministration of fresh vector to restore the transduced cell number. HAds recruit innate immune responses through a number of pathways. These viruses contain several pathogen-associated molecular patterns that are recognized by innate immune sensors or pattern-recognition receptors (PRRs) (reviewed in ref. 2Thaci B Ulasov IV Wainwright DA Lesniak MS The challenge for gene therapy: innate immune response to adenoviruses.Oncotarget. 2011; 2: 113-121Crossref PubMed Scopus (60) Google Scholar). The earliest events in the rapid recruitment of immunity were thought to derive from the binding of the capsid fiber protein to the coxsackie adenovirus receptor (CAR) and the interaction of the tripeptide Arg-Gly-Asp (RGD) motif within the capsid pentons with integrins on the host cell surface. The latter transmit signals through nuclear factor kappa B (NF-κB), resulting in the production of chemokines and cytokines, including interleukin 1 (IL-1). The work by Doronin et al. challenges the assumption that this interaction is the primary inductive factor and instead suggests that in fact it is TLR4 activation by the FX–hexon complex that provides the first innate response to FX-binding viruses. They demonstrated this effect by analyzing the genes that were differentially regulated in mice infected with FX-binding or non-binding HAds. The expression of 519 genes was increased in the spleen of mice 30 minutes after infection with wild-type (WT) virus. Because IL-1 is produced rapidly after infection, many of these genes might have been activated indirectly as a result of IL-1 signaling through the IL-1R–MyD88–NF-κB pathway. Therefore, to identify genes that were direct targets of HAds, the experiment was repeated in IL-1 receptor (IL-1R) knockout mice. This time expression of only 34 genes was increased, showing that IL-1-dependent feed-forward loops of transcriptional activation were indeed induced following initial activation of TLR4 during the early steps of HAd infection (Figure 1). Next, the differential effects of FX-binding ablated and WT viruses on these 34 genes were analyzed in IL-1R knockout mice. Using Ingenuity Pathway analysis, the investigators found that whereas NF-κB1, CREB1, and SRF pathways were activated after infection with both HAds, only the NF-κB1 pathway was differentially regulated between WT and FX-binding ablated virus, with the latter showing reduced levels of NF-κB1–related cytokines, including IL-1β. Because IL-1β transcriptional activation requires TRAF6, MyD88, and TRIF, which are signaling adapters for TLR activation, the authors next analyzed IL-1β production in TLR2, 4, 7, 8, and 9 knockout mice. Only TLR4−/− mice failed to produce IL-1β, demonstrating that a full-scale innate response to HAds requires TLR4–MyD88–NF-κB1 activity. Although these differences between the initial triggering events by WT and mutant viruses are important, the remaining pathogen-associated molecular patterns provided by hexon and fiber as well as the viral DNA and RNAs that activate internal PRRs such as TLR9 in endosomes, the DNA-dependent activator of interferon (IFN)-regulatory factors, and nucleotide oligomerization domain (NOD)-like receptors (NALP3 and NOD2) and RIG-I will ensure that subsequent innate triggering events occur irrespective of FX binding (Figure 1). As a consequence, both NF-κB1-dependent and NF-κB1-independent pathways will be activated, and cytokines and other inflammatory mediators will be produced, even in response to FX-binding-ablated virus. The multiplicity of Ad-associated stimuli for recruiting the innate immune system reduces the feasibility of strategies intended to ablate these complex interactions and thereby render adenovirus immunologically inert. Unfortunately, ablation of FX binding did not reduce targeting to macrophages and therefore does not prevent virus sequestration in the liver, but it is nonetheless possible that a reduction in the earliest and most potent signals by FX ablation may allow increased virus dosing with reduced toxicity, particularly if combined with strategies such as PEGylation, encapsulation in nanoparticles, or modifications to fiber to inhibit uptake by liver macrophages.3Prill J-M Espenlaub S Samen U Engler T Schmidt E Vetrini F et al.Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells.Mol Ther. 2011; 19: 83-92Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,4Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar It remains to be seen whether FX ablation alone will be able to reduce immediate adenoviral toxicity and immunogenicity to a degree that will justify reconsidering its use for gene replacement therapies. On the other hand, FX binding, with its link to the innate immune system, may be considered beneficial for the most widely used applications of adenoviruses in cancer therapy and vaccination because these usually seek to enhance the immune response. Paradoxically, therefore, the most important result of our new understanding of the interplay between FX, adenovirus, and innate immunity may be our ability to further increase, rather than to reduce, the proinflammatory environment the virus creates.