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OCR for page 67
6
Development of Therapeutics
H
istorical evidence suggests that, in response to an accidental or
intentional release of variola virus, smallpox vaccination would be
an effective public health measure to protect at-risk populations. To
be protective, however, vaccination must occur within 4 days of exposure
to the virus (Fenner et al., 1998; Mortimer, 2003). In addition, there are
contraindications to the administration of current smallpox vaccines, par-
ticularly among immunocompromised individuals. These individuals would
need alternative protective measures following exposure to variola virus.
To reduce the significant morbidity and mortality in cases of smallpox,
safe and effective therapeutic agents are required. By accelerating clearance
of the virus from ill individuals, such agents may also limit infectivity and
transmission of disease. Antiviral agents can also be useful for prophylaxis
after exposure has occurred. The availability of these agents has the poten-
tial to be important for both the treatment and prophylaxis of smallpox in
exposed persons identified after the 4-day period when vaccination is effec-
tive, and could be a valuable component of any effective control strategy. In
the last decade, substantial progress has been made in the development of
therapeutics with the potential to meet this need (see Tables 6-1 and 6-2).
However, these efforts have yet to yield an FDA-licensed agent for the treat-
ment or prevention of smallpox and other orthopoxviruses.
The 1999 IOM report identified the development of antiviral agents as
the most significant reason to retain stocks of live variola virus, primarily
because of the lack of availability of an effective therapeutic agent (either
currently or historically) that could serve as a standard for purposes of
comparison:
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LIVE VARIOLA VIRUS
The most compelling reason for long-term retention of live variola
virus stocks is their essential role in the identification and develop-
ment of antiviral agents for use in anticipation of a large outbreak
of smallpox. It must be emphasized that if the search for antiviral
agents with activity against live variola virus were to be continued,
additional public resources would be needed.
The 1999 report also suggested that having more than one antiviral
agent would be desirable because of the potential for the emergence of
drug-resistant variola strains. Replication-deficient forms of variola virus
could be used to develop new agents; ultimately, however, the live intact
virus would be required to ensure confidence in the results. The 1999 report
also noted that, given the lack of incentive for the development of smallpox
therapeutics in the private sector, significant public resources would need
to be mobilized.
This chapter reviews potential therapeutics for smallpox, regulatory
requirements for the development of such therapeutics, and the need for
live variola virus in this work.
POTENTIAL THERAPEUTICS FOR SMALLPOX
Potential therapeutics for smallpox include two drugs approved by the
FDA for other purposes, newly developed drugs, agents to block newly
identified poxvirus targets, and drugs that enhance or modulate the host’s
immune response.
Use of Drugs Approved by the FDA for Other Purposes
Because de novo drug development is an expensive and time-consuming
process (costing in excess of $500 million and requiring approximately
8–10 years of continuous effort) (Henderson and Fenner, 2001), the use of
licensed drugs approved for other purposes represents an attractive option
for antivirals against variola.
Cidofovir is a DNA polymerase inhibitor, licensed for the treatment of
cytomegalovirus-induced retinitis in HIV-infected individuals (Tesh et al.,
2004). Cidofovir also exhibits in vitro antiviral activity against poxviruses,
and is effective against cowpox and vaccinia virus infections in mice
(LeDuc et al., 2002; Baker et al., 2003; Quenelle et al., 2003; Magee et al.,
2005). Under an Investigational New Drug (IND) protocol from the FDA,
cidofovir can be used to treat acute smallpox and complications arising
from vaccinia infection when a patient has not responded to administra-
tion of vaccinia immune globulin (VIG) (LeDuc et al., 2002; reviewed in
Sliva and Schnierle, 2007). However, the utility of cidofovir for treating
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DEVELOPMENT OF THERAPEUTICS
smallpox is complicated by the fact that the drug is available only in a
topical or intravenous formulation. A topical formulation would have no
role in treating a systemic disease such as smallpox. Intravenous cidofovir
must be given as a 1-hour infusion in combination with multiple doses of
probenecid and requires sustained intravenous hydration and monitor-
ing of renal function. Even when given intravenously, the drug does not
cross the blood–brain barrier. Although cidofovir’s long half-life has the
advantage of allowing weekly dosing, problems with administration and
toxicity make large-scale use of this agent difficult. It is not likely to be
usable in resource-poor settings. The emergence of resistance is also a con-
cern because exposure of vaccinia to cidofovir resulted in the emergence
of mutations in the DNA polymerase gene, which is the target of the drug
(Becker et al., 2008).
Gleevec (also referred to as STI-571 or imatinib mesylate) is an FDA-
approved treatment for chronic myeloid leukemia that exhibits antiviral
activity against poxviruses. Gleevec blocks the action of Abl-family tyrosine
kinases (Druker et al., 1996) and thus blocks the egress of vaccinia virus
from infected cells in vitro (McFadden, 2005; Reeves et al., 2005; Yang et
al., 2005). It has also undergone in vitro testing against the monkeypox
and variola viruses with similar effects (Reeves et al., 2006). In addition,
Gleevec treatment promoted survival of mice following intranasal challenge
with vaccinia virus, and it has been suggested as a potential therapeutic for
postvaccination complications associated with vaccinia (Reeves et al., 2005).
The drug does not appear to interfere with the development of immunity
that protects against subsequent challenge. However, the protective benefit
of Gleevec was evident only at lower virus titers and only when the drug was
given less than 48 hours after exposure. Studies of Gleevec in rabbits infected
with rabbitpox and in mice infected with ectromelia showed much lower
antiviral activity than in other animal models (personal communication,
Dr. Daniel Kalman, Emory University, February 2009). The reduced activity
against higher titers of the inoculum virus, the requirement for administra-
tion shortly after inoculation, and the variable protection in poxvirus models
raise concerns about Gleevec’s potential for treating smallpox.
Newly Developed Therapeutics
To overcome the challenges associated with cidofovir discussed above,
orally bioavailable cidofovir derivatives have recently been developed
(HDP-cidofovir/CMX-001) (Ciesla et al., 2003; Buller et al., 2004; Kern
et al., 2004). CMX-001 also displays enhanced antiviral activity against
variola virus in comparison with cidofovir (Bradbury, 2002; Morris, 2002;
Sliva and Schnierle, 2007). The inhibitory activity of hexadecyloxypropyl-
CDV is 40–100 times grater than that of CDV in vitro in cells infected with
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TABLE 6-1 Development of Therapeutics for Smallpox
0
Drug Chemical Structure Mode of Action Reference
Cidofovira ATP analog that inhibits Baker et al., 2003; Magee et al.,
NH2
(CDV) DNA polymerase. When 2005, 2008; Krecmerová et al.,
incorporated into the 2007a
N X template strand, blocks DNA
elongation and 3′–5′ proof-
Y
N
O reading exonuclease activity.
Drug resistance seen in the
VV E9L gene (DNAP). FDA
OH
approved for cytomegalovirus
O P(O)(OH)2 retinitis in persons with HIV.
1, X = N, Y = CH
2, X = CH, Y = N
CDV CMX-001 Same target as CDV. Kern et al., 2002; Kern, 2003;
derivativesb,c: Esterification makes these Krecmerová et al., 2007b; Quenelle
NH2
CMX001 and derivatives more lipophilic et al., 2007b; Magee et al., 2008;
Table 6-1, Cidofovir (CDV)
HPMP-5-azaCb and increases uptake roughly Naesens et al., 2008; Parker et al.,
N 50-fold. CMX001 is 100-fold 2008
R01478
more active than CDV and
aw
NO does not produce renal
redrO n with vectors
toxicity.
O P OCH2CH2CH2OCH2(CH2) 14CH3
O-
Na+
HO
Table 6-1, CMX001
R01478
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vector, editable
HPMP-5-azaCb
NH2
N
N
O N
O
O PO
OR
STI-571 Blocks the Abl-family McFadden, 2005; Reeves et al., 2005
N
(Gleevec or tyrosine kinases needed for
imatinib the actin motility of
N
HN N
mesylate) intracellular viral particles
Table 6-1, HPMP-5-azaC (IMV), thus blocking egress
CH3
of IMV from cells. FDA
R01478
approved for chronic myeloid
redrawn as vectors leukemia.
HN
CH3
O N
N
Table 6-1, STI-571
R01478
continued
redrawn as vector
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TABLE 6-1 Continued
Drug Chemical Structure Mode of Action Reference
ST-246d Inhibits virus release by Yang et al., 2005; Quenelle et al.,
H
targeting a pox protein (p37 2007a,b
HH or 60L for cowpox or F13L
H for vaccinia) that is essential
O
for envelopment of IMV.
N
O
O HN
F
F F
Table 6-1, ST-246
R01478
redrawn
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Targets the Thymidine Kinase Kern et al., 2009
4′-thioIDUe O
(TK) gene to inhibit DNA
R
synthesis.
NH
N O
YO
SX
OY
aCidofovir {HPMPC, CDV, 1-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl] cytosine}.
b{1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine; HPMP-5-azaC} is an analog of CDV.
cCMX001 is hexadecyloxypropyl CDV and has much better oral bioavailability than CDV.
dST-246 [N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-di oxo-4,6-etheneocycloprop[f]isoindol-2(1H)-yl)-benzamide] used at 20 µg/ml in cell cultures re-
duces EMV by 6 logs and reduces IMV by 2 logs.
e1-(2′-deoxy-4′le 6-1, 4’-thioIDU
Tab-thio-β-D-ribofuranosyl)-5-iodouracil (4′-thioIDU). The R can be F, Br, I, CH3, CF3, or a phenyl group. The X can be H or OH. The
Y can be H or an acetyl group.
R01478
redrawn as vectors
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LIVE VARIOLA VIRUS
TABLE 6-2 Clinical Aspects of Therapeutic Agents for Smallpox in
Humans
Dose and Route of
Drug Administration Issues
Cidofovir 5 mg/kg given intravenously Severe renal toxicity; co-administration of
weekly/biweekly; also used probenecid necessary; hydration
topically and intralesionally requirement; used off label for recurrent
laryngeal papillomatosis, molluscum
contagiosum, and human papillomavirus
Esters of Dose not yet defined; taken Phase I and II human clinical trials under
Cidofovir orally way; no reported renal toxicity to date;
(CMX001) higher bioavailability than CDV
Gleevec 400–800 mg/day; taken Chemotherapeutic agent; side effects include
orally edema, cytopenia, and hepatotoxicity
ST-246 500–2000 mg/day; taken Minimal toxicity seen in human dosing
orally for 14 days; other trials
routes of administration
(intravenous, liquid
suspension) being considered
variola, cowpox, vaccinia, or ectromelia virus. Protection of mice from
lethal mousepox infection has been demonstrated (Parker et al., 2008), and
CMX-001 was effective against mousepox in the C57BL/6 strain, which
is considered to have a course of infection more similar to that of variola
than its progression in other mousepox strains when given 4 days after
inoculation (Parker et al., 2009). Other derivatives of cidofovir could prove
effective as well (Lebeau et al., 2006; Stittelaar et al., 2006; Hostetler et
al., 2007; Hostetler, 2009). Orally bioavailable cidofovir derivatives have
shown negligible renal toxicity, a significant advantage over the intravenous
formulation. CMX-001 has been given to a patient with eczema vaccinatum
who did not respond to ST-246 (CDC, 2009). A recently completed human
volunteer phase I multidose study with more than 100 subjects demon-
strated no significant adverse events, and phase II trials are being initiated
(Painter and Hostetler, 2004; Ruiz et al., 2007).
ST-246, which was discovered from a high-throughput screen of 356,240
small-molecule inhibitors of vaccinia virus replication, is currently being
used in human trials. This antiviral drug targets the vaccinia virus protein
F13, which is essential for envelopment and egress of the intracellular
mature virions (MV) and subsequent viral spread (Yang et al., 2005). Cell
cultures infected with six different variola isolates or seven different monkey-
pox isolates showed reduced cytopathic effects, virus production, and comet
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DEVELOPMENT OF THERAPEUTICS
tail formation after treatment with nanomolar amounts of ST-246. ST-246
is 8,000 times more potent than cidofovir in vitro against poxviruses, is
orally bioavailable, and is stable at room temperature. It has proven to be
effective in blocking replication of all orthopoxviruses that have been tested
in vitro (Duraffour et al., 2007) and in protecting mice (Yang et al., 2005;
Quenelle et al., 2007a), rabbits (Nalca et al., 2008), and ground squirrels
(Sbrana et al., 2007) from orthopoxvirus challenge. Animals infected with
monkeypox, cowpox, ectromelia, and variola viruses that received ST-246
were protected from lethal infection and also mounted a protective immune
response (Bolken and Hruby, 2008; Nalca et al., 2008). ST-246 in combina-
tion with CMX-001 displays synergistic antiviral effects against vaccinia
and cowpox in animals without increasing toxicity (Quenelle et al., 2007b;
Whitley, 2008). In 2007, a 14-day course of ST-246 was used in conjunction
with cidofovir and VIG under an emergency IND to treat a severe case of
eczema vaccinatum in an infant who was infected with vaccinia as a result
of contact transmission (Vora et al., 2008). Since cidofovir and VIG were co-
administered with ST-246, however, it is not clear that the resolution of the
infection is attributable entirely or even partially to ST-246. Human phase
I trials of ST-246 have been completed. The drug was given to 31 healthy
individuals in a single dose ranging from 500 mg to 2000 mg daily in a
fasting and nonfasting state, with an 8-person placebo group used for com-
parison (Jordan et al., 2008). Side effects were minimal, and only reversible
neutropenia was seen more often in the treated than in the placebo group.
Important information on ST-246 has been obtained: the variola gene
product targeted by ST-246 is known, and the doses have been shown to
be effective against poxviruses in mice and nonhuman primates. However,
clinical data are needed on the use of ST-246 in humans; studies to provide
these data are under development for naturally occurring human monkey-
pox but will be difficult to implement and monitor. An important caveat for
antiviral drugs such as ST-246 that exhibit high potency in vitro is that they
can be tested only in model systems or against other poxvirus infections
in humans, and it is impossible to know with certainty how they would
perform against smallpox in the event of its reemergence.
Work on the development of new drugs has also continued in Russia.
VECTOR reports having conducted screening of more than 5,000 chemi-
cal compounds for their antiviral activity, and about 80 compounds
active against surrogate orthopoxviruses (vaccinia virus, cowpox virus,
and ectromelia virus) are said to have been identified. In testing done in
cell culture, VECTOR reports that 60 compounds demonstrated antiviral
activity against variola virus (Zakirova et al., 2004; Ivanov et al., 2005,
2008; personal communication, Ilya Drozdov, WHOCC for Orthopoxvirus
Diagnosis and Repository for Variola Virus Strains and DNA, March 27,
2009).
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LIVE VARIOLA VIRUS
Agents to Block Newly Identified Poxvirus Targets
Further research on poxvirus replication has made it possible to identify
possible poxvirus drug targets, and assessment of molecules blocking these
targets has begun (Yang et al., 2005; Sliva and Schnierle, 2007; Tse-Dinh,
2008). Three enzymes involved in vaccinia virus replication have been iden-
tified and crystallized: thymidine kinase (TK), deoxyuridine triphosphatase
(DUTPase), and uracil DNA glycosylase (UDG) (Whitley, 2008). DNA
polymerase nucleoside inhibitors (Fan et al., 2006; Prichard et al., 2007),
nucleoside inhibitors of S-adenosyl-L-homocysteine hydrolase (De Clerq
and Holy, 2005; Roy et al., 2005; Yang and Schneller, 2005; Arumugham
et al., 2006), targets of topoisomerase I (Da Fonseca and Moss, 2003;
Bond et al., 2006; Fujimoto et al., 2006; Perry et al., 2006), and other
egress inhibitors (Bailey et al., 2007) have been evaluated for their ability
to block poxvirus replication. It has been suggested that the new 4′ thioIDU
(TK inhibitor) might be an additional component of combination therapy
since it can block replication of CMX–001- and ST-246-resistant mutants
(Kern et al., 2009). The use of combination antiviral therapy is favored as
it may slow the development of drug-resistant strains of variola and other
orthopoxviruses. When administered intraperitoneally or orally, 4′ thioIDU
was shown to be protective against both cowpox and vaccinia in mice (Kern
et al., 2009). Selectivity indices (CC50/EC50) ranged from more than 200
to 2,000 for 4′ thioIDU; in contrast, the values for CDV were more than 9
to more than 32 (Kern et al., 2009). However, 4′ thioIDU, like CDV and
its derivatives, is toxic for dividing cells.
Agents That Enhance or Modulate the Host Immune Response
Enhancing or modulating the host immune response is an alterna-
tive or adjunctive therapeutic approach to controlling smallpox through
antiviral drugs that disrupt the replication cycle. Providing passive immu-
nity through the transfer of protective antibodies from an immune to
a susceptible individual can lend temporary, but potentially life-saving,
protection.
As an example, this approach was used therapeutically in the 1940s in
Morocco. Antiserum was obtained from smallpox survivors soon after the
last scabs fell off, and was then administered to newly arriving patients at
the clinic in doses of 10–20 ml per day (Couzi and Kircher, 1941). Among
the 200 persons given this treatment, including 75 patients with advanced
hemorrhagic disease, all survived. However, this was a report of clinical
experience, not a controlled study, and use of passive antibodies as therapy
for clinically evident, established infection has not been demonstrated to be
effective against systemic viral illnesses.
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DEVELOPMENT OF THERAPEUTICS
Today, VIG collected from individuals with high antibody titers from
repeated immunization is given to confer passive immunity in individuals
with complications resulting from smallpox vaccination, and is the only cur-
rently available intervention other than unlicensed antiviral drugs (Kempe
et al., 1961; Wittek, 2006). Two intravenous formulations of VIG (Cangene
and Dynport) have been licensed by the FDA for the management of
patients with progressive vaccinia, eczema vaccinatum, severe generalized
vaccinia, and extensive body surface involvement or periocular implanta-
tion of vaccinia following inadvertent inoculation (Wittek, 2006). When
given to exposed individuals, VIG is expected to provide protection against
infection for approximately 2–3 weeks, presumably through its neutralizing
activity against vaccinia.
The conserved orthopox protein vaccinia B5/variola B6 is a major
neutralizing target for VIG, although major neutralizing sites on B5 are
exposed differently on the variola ortholog (Aldaz-Carroll et al., 2007). B5
is needed to wrap the MV to form extracellular virus, and interactions with
actin are necessary for virion egress from the infected cell (see Aldaz-Carroll
et al., 2005, 2007).
More recently, humanized chimpanzee monoclonal antibodies specific
for the B5 and A33 envelope glycoproteins of vaccinia virus and the variola
virus homologs have been reported to inhibit the spread of vaccinia and
variola viruses in vitro and have conferred protection in a mouse model
of poxvirus infection (Chen and Ron, 2006; Chen et al., 2007). These
antibodies may be useful for treating vaccine-related complications or for
prophylaxis or therapy of smallpox.
VECTOR reports that since 2002 it has been working to develop human
recombinant antibodies as therapeutics for treatment of smallpox infection
(Tikunova et al., 2005; Yun et al., 2006; Dubrovskaia et al., 2007). To that
end, a panel of 66 unique human mini-antibodies against orthopoxviruses,
including variola virus, was selected from VECTOR’s combinatory phage
library and from that obtained from The Medical Research Council (UK).
Half of the antibodies selected were tested for their ability to neutralize
variola virus. Based on the most promising antibodies, VECTOR states that
four fully human antibodies against variola virus were constructed, their
affinity constants were measured, and they were tested for their ability to
neutralize vaccinia virus (personal communication, Ilya Drozdov, WHOCC
for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and
DNA, March 27, 2009).
Other antiviral drugs, such as ribavirin, that are not poxvirus specific
but counteract host responses represent another therapeutic approach to
smallpox infection (Baker et al., 2003). The lower specificity and poten-
tial toxicity of such drugs make them less ideal, but some have been
powerful modulators of disease severity with life-saving effects. Two recent
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LIVE VARIOLA VIRUS
reviews have suggested that agents such as tumor necrosis factor (TNF)
inhibitors that are used to treat septic shock may be effective (Harrison et
al., 2004; Jahrling et al., 2005). However, recent transcriptome profiling
studies (Rubins et al., 2004) have shown that virulent poxvirus infection
in primates appears to suppress TNF expression, raising concerns that
further TNF suppression may enhance virulence and produce more severe
disease. Moreover, several laboratory studies have suggested that the TNF-
inhibiting genes of the poxviruses are a crucial part of pathogenesis (Sedger
et al., 2006; Bartee et al., 2009). TNF production therefore is likely to be a
protective mechanism counteracted by orthopoxvirus proteins. This finding
also suggests that disease would be exacerbated by anti-TNF treatment.
As noted, postexposure smallpox vaccination is beneficial if given
shortly after the contact. In addition to accelerating the development of
specific antiviral responses, vaccinia inoculation may elicit immediate innate
responses that control the initial progression of infection and modulate
disease severity. In a monkeypox model, however, postexposure vaccination
was not as effective as cidofovir or its derivatives (Stittelaar et al., 2006).
Summary
At present, two drugs that are FDA-approved for other purposes—
cidofovir, a DNA synthesis inhibitor, and Gleevec, a tyrosine kinase
inhibitor—hold potential for use as therapeutics against smallpox. Cidofovir
can be used on an investigational basis for treating severe orthopoxvirus
infections, including smallpox. FDA-approved preparations of VIG are also
available. New drugs that are under evaluation and show promise include
orally bioavailable esters of cidofovir (CMX-001) and ST-246, an inhibitor
of virus egress. ST-246 has been given to human volunteers and has been
administered on a compassionate use therapeutic basis to a 2-year-old child
with eczema vaccinatum following vaccinia exposure. New types of VIG
are also being developed that target specific proteins such as variola B6R.
REgULATORy REqUIREMENTS
Recommendations and requirements for U.S. licensure of drugs intended
for the prevention and treatment of variola infection are outlined exten-
sively in a 2007 Guidance for Industry document prepared by the FDA (see
also Chapter 1) (FDA, 2007). The guidance pertains primarily to small-
molecule therapeutics, although its main principles can also be applied to
biological products such as immunoglobulin preparations, monoclonal anti-
bodies, and therapeutic proteins. Of particular relevance, demonstration
of efficacy against live variola virus appears to be an essential step on the
pathway to licensure (see Table 6-3). More specifically, use of the Animal
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DEVELOPMENT OF THERAPEUTICS
TABLE 6-3 Scientific Pathway for Drug Development
Steps Assays Criteria
1. Rational Design Computerized displays of viral Drug fits target
(optional) proteins and best fit of drugs
2. Cell culture tests Drug effects on cytotoxicity, Efficacy/toxicity (EC50/CC50)
of effects of drugs virus production, cytopathy, >10
on infected cells comet formation, generation of
resistant mutants
3. Small-animal Use of mice infected with Doses and routes for treatment
model ectromelia, cowpox, or found where virus titers decrease
vaccinia for initial studies of by >3 logs, disease signs are
drug safety and efficacy in vivo eliminated in most animals, and
mortality decreases >50%
4. Large-animal Cynomolgus macaques given Same as above
model monkeypox intratracheally or
variola intravenously should be
tested for shedding, virus
titers, disease signs
or
A nonrodent model, such as
one using rabbits or monkeys,
should be tested for shedding,
virus titers, transmission,
disease signs
• Phase I/II clinical trials
5. Human beings Safety trials in humans should
• Treatment or emergency use
monitor blood chemistries and
other biomarkers for toxicity; Investigational New Drug
infected people should also be (IND) application for severe
tested for virus vaccinia or other
orthopoxvirus infections
• Emergency Use Authorization
(see Chapter 1)
Rule (see Chapter 1) or any other currently available regulatory pathway to
achieve licensure is essentially precluded by the exceptionally narrow host
range of variola virus; the lack of any previously recognized effective drug
for use in head-to-head comparison with any new compound; and known
and possible differences between variola and other orthopoxviruses in dis-
ease characteristics, drug susceptibility, and host range (Jordan and Hruby,
2006; Bolken and Hruby, 2008). Further, FDA officials have highlighted the
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0 LIVE VARIOLA VIRUS
critical importance of conducting safety studies in normal human volunteers
and potentially in patients with underlying medical conditions. The FDA
also recommends studies using animal models that mimic human disease
progression to provide supporting evidence of clinical efficacy (Roberts et
al., 2008), but the development of a nonhuman primate challenge model for
variola has been extraordinarily difficult in practice (see Chapter 4); more-
over, some orally administered candidates (e.g., CMX-001) are not absorbed
in these animals. While data derived from studies of other orthopoxviruses
(e.g., monkeypox or vaccinia) cannot be considered definitive evidence of
antivariola activity, the FDA guidance indicates that exploratory studies
with these viruses can provide important adjunctive information.
In addition to variola-specific considerations, general considerations
applicable to the licensure of any antiviral agent include analysis of in vitro
activity in conjunction with other drug candidates, selection and evaluation
of resistant viral strains, and consideration of drug–vaccine and drug–drug
interactions.
NEED FOR LIVE VARIOLA VIRUS
Fewer than 10 percent of published studies related to the development of
therapeutics for smallpox have actually involved the use of live variola virus.
This fact demonstrates that much can be accomplished by other means. For
both scientific and regulatory reasons, however, the advanced stages of drug
development will require evaluations involving live variola virus.
In the 30 years since the eradication of smallpox, variola stocks have
been used to complete the sequence of at least 49 VARV isolates (see Chap-
ter 5) (Esposito et al., 2006), to gain some understanding of the genetic
differences between virulent and nonvirulent poxviruses, to understand the
neutralizing epitopes that could be targeted by VIG, to further understand
the replication cycle of variola in order to identify potential targets for
antiviral agents, and to design and evaluate potential variola model chal-
lenge systems for purposes of confirming the efficacy of candidate antiviral
agents under the Animal Rule.
Although preliminary testing of antivirals can use related orthopox-
viruses, live variola virus should be used in cell culture as the ultimate test.
Host cell responses that define the course of variola infection in cell cul-
ture or in vivo, such as changes in gene expression or changes in signaling
pathways, miRNA, or secreted cytokines, should be investigated to identify
networks of responses that could serve as biomarkers of inhibition of virus
infection by a candidate drug. Host responses to similar viruses, such as
monkeypox or vaccinia, could be used to identify biomarkers associated
with virulent or benign infection. The changes in these profiles found to be
associated with successful drug treatment in these models could be used as
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DEVELOPMENT OF THERAPEUTICS
candidate biomarkers indicating poxvirus control and further evaluated in
nonhuman primates infected with variola. Similarly, the pharmacokinetic
and pharmacodynamic properties of the drug in these models need to reflect
those in humans.
If such biological parameters can be validated, it may eventually be
possible to use these measures in developing an alternative to testing with
live variola virus. For example, a VIG formulation containing monoclonal
antibodies to variola B6 could be tested in mice infected with a vaccinia
virus expressing the orthologous vaccinia B5. An ectromelia infection of
mice could perhaps have a profile similar to a variola infection of mice,
resulting in the same alterations in host response whether the challenge
virus was ectromelia or variola. Thus, host responses to drug treatments
after ectromelia infection could serve as surrogate biomarkers for efficacy
in the absence of live variola infection. Nevertheless, in accordance with
Table 4-1 in Chapter 4, biomarkers developed in nonhuman primate models
would be more likely to reflect the disease progression in human beings and
therefore make better surrogates for disease progression in antiviral testing
studies. Any predictions about drug activity against variola would have to
be made with great caution.
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