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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.
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