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9 Discovery Research T he combination of recent technological advances in molecular biology, genomics, and computational biology, coupled with the intimate relationship between variola virus and the human immune system in particular, creates unusual opportunities for scientific discovery. The guiding rationale for work with variola is the possibility of obtain- ing novel insights that would lead to new smallpox prevention strategies, diagnostic approaches, and therapeutic interventions. Given that variola exclusively infects humans under natural conditions and has adapted to specifically modulate the human immune system, much could be learned about human biology from studies with this virus. As variola proteins that dampen or manipulate a particular immune response are identified, these viral proteins, or portions thereof, become candidate novel therapeutics for autoimmune or inflammatory diseases in which the host response is aber- rant or overactive. The 1999 IOM committee offered two conclusions related to discovery research: • Live or replication-defective variola virus would be needed if studies of variola pathogenesis were to be undertaken to provide information about the response of the human immune system. • Variola virus proteins have potential as reagents in studies of human immunology. Live variola virus would be needed for this purpose only until sufficient variola isolates had been cloned and sequenced. 

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 LIVE VARIOLA VIRUS The 1999 IOM committee acknowledged that variola virus plays a unique role in contributing to understanding of the human immune system. Research in this area could yield further information about human-specific reagents with therapeutic or immunomodulatory potential. This chapter examines opportunities for discovery research involv- ing variola virus in three areas: the potential to gain new insights into the pathogenesis of smallpox through the capabilities offered by systems biology, understanding of the subversion and modulation of human immune responses, and the possibilities for development of novel variola-based therapeutics. The final section addresses the need for live variola virus to conduct this work. SySTEMS BIOLOgy AND SMALLPOX PATHOgENESIS While some progress has been made since 1999 toward elucidating the pathogenesis of smallpox and characterizing viral immunomodulatory activities, much more remains to be learned. The synthesis of molecular biology, genomics, and computational biology, or “systems biology,” offers promising approaches for understanding smallpox pathogenesis, human immunology, and other aspects of host defense and for identifying novel therapeutic targets and strategies. Systems biology refers to the study of the behavior of complex bio- logical organization and processes in terms of the molecular constituents (Kirschner, 2005). It is made possible by the availability of broad-based, genome-wide, high-throughput approaches for measuring the abundance and localization of DNA, RNA, and protein and their interactions within an entire biological system. Although in its early days, this discipline offers the promise of revealing rules and features that can lead to predictions about the vulnerabilities and control points of a cell or an organism. For instance, this approach could be used to examine the interaction between variola and an infected target cell, or the broader interactions between variola and an infected host as in studies by Rubins and colleagues (2004) (see below), but with a more complete set of measurements of RNA and protein. The use of systems biology techniques in a more comprehensive and integrated fashion represents a largely untapped resource for learning more about the variola life cycle and the interactions between variola virus and its host. Limited studies of pathogenesis have been performed with variola virus in nonhuman primates. Jahrling and colleagues (2004) describe a model of lethal disease in cynomolgus macaques with features of late, severe small- pox, achieved using high intravenous doses of variola virus (the Harper and India 7124 strains). DNA microarrays were performed in peripheral blood cells from these infected animals to examine host gene expression

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 DISCOVERY RESEARCH patterns (Rubins et al., 2004) (see Chapter 4). From these data, groups of genes were identified, as well as coregulated biological processes associated with these genes and their products. Some of these processes, such as “cell proliferation,” had not been emphasized previously as a prominent feature of variola infection of primates. Although these studies revealed important information regarding smallpox pathogenesis—such as the prominence of an interferon-associated response; unusual suppression of the NFkB response system; and the possible importance of other biological processes, such as cell proliferation—they were limited in a number of ways. First, the nonhuman primate model was inadequate for studying early aspects of smallpox as it occurred naturally in humans. In addition, the kinds of measurements performed in these studies were limited and did not include the newly discovered and critical noncoding RNAs of primates, or genome- wide patterns of protein expression, or the interactions of proteins and nucleic acids, all of which can now be quantified using high-throughput genome-wide technologies. Furthermore, the responses of different indi- vidual cell types have not yet been explored, even though it is clear that distinct biology is found in different cells. With today’s improved high-level containment research facilities and more powerful research technologies, a great deal more might be learned about variola–host interactions, with relevance to the development of smallpox therapeutics. Poxviruses replicate in the cytoplasm of susceptible host cells and con- tain regulatory sequence elements that are virus specific (Moss, 1996). An accurate functional analysis of poxvirus proteins may require expression systems that replicate the posttranslational modifications found in naturally infected cells and hosts, and may not be possible with typical protein expres- sion systems (e.g., bacteria, yeast, or insect cells). Moreover, some viral pro- teins may have multiple, unrelated functions or may function primarily as a complex with other viral or host cell proteins, and thus may be biologically inert if expressed in the wrong cell type or in the absence of a productive infection. Bearing this in mind, it is possible that some variola proteins will require analysis in the context of live infection of human cells or through coexpression experiments with a number of other viral protein partners. SUBVERSION AND MODULATION OF HUMAN IMMUNE RESPONSES At the time of the 1999 IOM report, it was known that poxviruses encode the largest number of putative immunomodulatory proteins of any group of mammalian viruses (Barry and McFadden, 1997). As of this writing, only five putative immunomodulatory proteins from variola virus have been characterized: D12/SPICE (smallpox inhibitor of complement enzymes) (Liszewski et al., 2008), G3R/CKBP-II (variola virus high-affinity

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 LIVE VARIOLA VIRUS secreted chemokine-binding protein type II) (Smith et al., 1997), B9R (an IFN-g inhibitor) (Seregin et al., 1996), G2R (a tumor necrosis factor inhibi- tor) (Alejo et al., 2006), and D5L (an IL-18-binding protein) (Esteban et al., 2004). The findings from this work, although limited, suggest that variola proteins have potent biological activity and may have special value in blunting human immune responses. Some of these findings are summa- rized below to illustrate the possible rewards of future work on these and other variola proteins, which could also yield insights into the mechanisms of variola pathogenesis. Variola CrmB encodes a tumor necrosis factor receptor (TNFR) homo- logue that acts as a soluble decoy of TNFR, as well as a chemokine inhibi- tor through its C terminal domain—the smallpox virus-encoded chemokine receptor (SECRET) domain (Alejo et al., 2006). This is the first example of a dual function for a poxvirus decoy molecule. The SECRET domain was subsequently identified in another variola TNFR homologue (CrmD) and three other orthopoxvirus-encoded secreted proteins (Alejo et al., 2006). Both variola and ectromelia virus encode soluble decoys that inhibit the activity of IL-18, an important proinflammatory cytokine (Esteban et al., 2004). Using surface plasmon resonance, it has been shown that both pro- teins have higher affinity for murine than for human IL-18, which is similar to human IL-18BP and an ortholog encoded by molluscum contagiosum virus (Xiang and Moss, 1999). Variola IL-18-binding protein (IL-18BP) also binds to glycoaminoglycans, whereas the ectomelia ortholog does not (Esteban et al., 2004). The 2.0-Å resolution crystal structure of a binary complex human IL-18 and ectromelia IL-18BP was recently solved (Krumm et al., 2008), and reveals significant conformational changes at the binding interface. The residues of ectromelia IL-18BP at the interface are conserved in both human IL-18BP and viral homologues. Although functional analysis of related immunomodulatory proteins from other orthopoxviruses can provide insight into the activities of their variola-encoded counterparts, this approach may not always provide an accurate understanding of the virulence factors of variola. For instance, direct comparison of the variola-encoded complement inhibitor SPICE with similar evasion proteins encoded by vaccinia (VCP) and monkeypox (MOPICE) revealed that VCP and MOPICE were approximately 100-fold less efficient than SPICE (Liszewski et al., 2006). This work suggests that studies involving viral gene products from even closely related orthopox- viruses will not necessarily provide the same information that would be attained by directly examining variola virulence proteins and immune eva- sion proteins. A relatively restricted number of variola proteins have been studied in detail, and although this work represents an important step forward, much remains to be learned about these and other variola gene products.

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 DISCOVERY RESEARCH The 1999 IOM report suggested that it would be possible to study variola protein products in isolation once a number of virus isolates had been sequenced. However, given the regulatory hurdles facing the use of variola-specific gene sequences and the restrictions associated with the use of live variola virus, most studies on poxvirus immune evasion have been performed with related orthopoxviruses instead of variola. More than a dozen predicted immunomodulatory proteins encoded by variola major have yet to be fully tested and characterized (McFadden, 2004). Recent studies conducted with related orthopoxviruses, such as cowpox and monkeypox, have revealed previously unrealized immune evasion/ subversion mechanisms that may be relevant to smallpox pathogenesis. For instance, cowpox expresses proteins that downregulate MHC Class I molecules on the infected cell surface (Byun et al., 2007; Dasgupta et al., 2007). By reducing MHC Class I expression, the virus is able to evade rec- ognition by cytolytic CD8+ T cells in a manner similar to the evasion strate- gies employed by many herpesviruses. Monkeypox has developed an even more intriguing strategy of host immune system manipulation by triggering a nonresponsive state in either CD4+ or CD8+ T cells that come into direct contact with monkeypox-infected monocytes (Hammarlund et al., 2008). These studies were performed by infecting primary human peripheral blood monocytes and measuring cytokine production by poxvirus-specific T cells using intracellular cytokine staining analysis. It is not known whether vari- ola expresses similar or possibly an even more extensive battery of immuno- modulatory genes that could directly block human T cell recognition and/or antiviral function in similar in vitro experiments. Also unknown is whether different strains of variola major and variola minor differ with respect to their ability to evade host T cell responses. Variations in the expression of various immunomodulatory proteins could explain the dramatic differences in pathogenesis and mortality rates that are associated with these two forms of smallpox, as well as with different strains of variola major. In addition to evading host T cell responses, poxviruses are known to subvert antiviral innate immune responses, including the attenuation of type I interferon, proinflammatory cytokines, and chemokine production. Upon infection, viral nucleic acids can be sensed through a variety of path- ways by host immune cells to trigger an immune response. Toll-like recep- tor (TLR)3, TLR7, and TLR9 are endosomal TLRs that recognize dsRNA, ssRNA, and viral DNA. RIG-I and MDA-5 are cytosolic RNA sensors (Kawai and Akira, 2008). Signaling through these pathways leads to type I interferon production and NF-κB activation. AIM2 is a recently identified cytosolic DNA sensor that may link DNA virus infection to inflammasome activation (Hornung et al., 2009). How poxviruses are sensed in vari- ous immune cells has just begun to be understood. Ectromelia virus (the causative agent of mousepox) activates pDCs through TLR9, and mice

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 LIVE VARIOLA VIRUS lacking TLR9 are more susceptible to ectromelia infection (Samuelsson et al., 2008). Infection of murine keratinocytes with vaccinia virus containing a deletion of the immunomodulatory gene E3L triggers a vigorous innate immune response that is dependent on cytosolic RNA sensing pathway and transcription factor IRF3 (Deng et al., 2008). Human macrophages sense myxoma virus and produce type I interferon and TNF-α that is dependent on RIG-I and IRF3 (Wang et al., 2008). Overall, then, poxviruses can be sensed by different pathways in a variety of immune cells to trigger an anti- viral response, but there is a relative dearth of information about variola virus and its specific interactions with the human innate immune system. NOVEL VARIOLA-BASED THERAPEUTICS Genome sequences from 45 strains of variola currently provide a set of diverse variola-specific proteins and variants that might be expressed and screened for biological activities of interest. These proteins could themselves serve as immunomodulatory agents or might provide leads for the develop- ment of related molecules. Additional variola genome sequences from as yet uncharacterized variola isolates might be expected to yield new sequence variants and expand this set of potential novel biologicals. It should be noted that specialized expression systems may be necessary for critical spe- cific posttranslational modifications of these proteins. Moreover, some viral proteins may have multiple, unrelated functions or may function primarily in a complex or in concert with other viral or host cell proteins, and thus may fail to demonstrate the relevant phenotype if expressed in the wrong cell type or in the absence of a productive variola virus infection. Many viral immunomodulatory proteins act with high specificity against a particular immune function or pathway and do so at very low doses (femtomolar to nanomolar), making these proteins potentially fea- sible for use as therapeutics to treat diseases of overactive immune function or inflammation (McFadden and Murphy, 2000; Shisler and Moss, 2001; Johnston and McFadden, 2003; Seet et al., 2003). The field of virogenomics (Fruh et al., 2001; Kellam, 2001; DeFilippis et al., 2003) is emerging as a means of future drug discovery and will continue to flourish as knowledge and understanding of host–pathogen interactions increases. NEED FOR LIVE VARIOLA VIRUS Comparative studies of variola major and variola minor in primary human cells have not been performed. The differences in virulence between the two or among different strains of variola major may lie in these inter- actions. Because variola was eradicated prior to the marked advances in modern cellular immunology and molecular biology techniques that have

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 DISCOVERY RESEARCH since occurred, understanding of human immune responses to variola infec- tion remains very limited. Live variola virus would be required to perform these comparative in vitro studies. Live virus would also be needed for the use of systems biology approaches in an improved nonhuman primate model with the goal of identifying novel therapeutic targets. With more than 40 strains of variola now having been sequenced, there is ample opportunity to study specific variola proteins, and thereby advance understanding of host–pathogen interactions and develop potential new therapeutic drugs. Live virus would be useful for initiating some of these studies but would not be required for most of this research. REFERENCES Alejo, A., M. B. Ruiz-Argüello, Y. Ho, V. P. Smith, M. Saraiva, and A. Alcami. 2006. A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus. Proceedings of the National Academy of Sciences of the United States of America 103:5995–6000. Barry, M., and G. McFadden. 1997. Virokines and viroceptors. In Cytokines in health and disease, edited by D. G. Remick and J. S. Friedland. New York: Marcel Dekker. Byun, M., X. Wang, M. Pak, T. H. Hansen, and W. M. Yokoyama. 2007. Cowpox virus exploits the endoplasmic reticulum retention pathway to inhibit MHC class I transport to the cell surface. Cell Host Microbe 2(5):306. Dasgupta, A., E. Hammarlund, M. K. Slifka, and K. Früh. 2007. Cowpox virus evades CTL recognition and inhibits the intracellular transport of MHC class I molecules. Journal of Immunology 178:1654. DeFilippis, V., C. Raggo, A. Moses, and K. Früh. 2003. Functional genomics in virology and antiviral drug discovery. Trends in Biotechnology 21(10):452–457. Deng, L., P. Dai, T. Parikh, H. Cao, V. Bhjoy, Q. Sun, Z. J. Chen, T. Merghoub, A. Houghton, and S. Shuman. 2008. Vaccinia virus subverts a mitochondrial antiviral signaling protein- dependent innate immune response in keratinocytes through its dsRNA binding protein E3. Journal of Virology 82:10735–10746. Esteban, D. J., A. A. Nuara, and M. L. Buller. 2004. Interleukin-18 and glycosaminoglycan binding by a protein encoded by variola virus. Journal of General Virology 85(Pt. 5):1291–1299. Fruh, K., K. Simmen, B. G. Luukkonen, Y. C. Bell, and P. Ghazal. 2001. Virogenomics: A novel approach to antiviral drug discovery. Drug Discovery Today 6 (12):621–627. Hammarlund, E., A. Dasgupta, C. Pinilla, P. Norori, K. Früh, and M. K. Slifka. 2008. Monkey- pox virus evades antiviral CD4+ and CD8+ T cell responses by suppressing cognate T cell activation. Proceedings of the National Academy of Sciences of the United States of America 105:14567. Hornung, V., A. Ablasser, M. Charrel-Dennis, F. Bauernfeind, G. Horvath, D. R. Caffrey, E. Latz, and K. A. Fitzgerald. 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase- 1-activating inflammasome with ASC. Nature 458(7237):514–518. Jahrling, P. B., L. E. Hensley, M. J. Martinez, J. W. LeDuc, K. H. Rubins, D. A. Relman, and J. W. Huggins. 2004. Variola virus infection of cynomolgus macaques: A model for human smallpox. Proceedings of the National Academy of Sciences of the United States of America 101:15196–15200. Johnston, J. B., and G. McFadden. 2003. Poxvirus immunomodulatory strategies: Current perspectives. Journal of Virology 77(11):6093–6100.

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