discovery of antivirals against smallpox · virus (smallpox virus) and molluscum contagiosum virus,...

Discovery of antivirals against smallpoxStephen C. Harrisona,b, Bruce Albertsc, Ellie Ehrenfeldd, Lynn Enquiste, Harvey Finebergf, Steven L. McKnightg,Bernard Mossh, Michael O’Donnelli, Hidde Ploeghj, Sandra L. Schmidk, K. Peter Walterl, and Julie Theriotm

aHarvard Medical School, Howard Hughes Medical Institute, Seeley Mudd Building, Room 130, 250 Longwood Avenue, Boston,MA 02115; cNational Academy of Sciences, 2101 Constitution Avenue, NW, Washington, DC 20418; dLaboratory of Infectious Disease,National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 50, Room 6120, 50 South Drive, Bethesda,MD 20892; ePrinceton University, 314 Schultz Laboratory, Washington Road, Princeton, NJ 08544; fInstitute of Medicine, 2101Constitution Avenue, NW, Washington, DC 20418; gDepartment of Biochemistry, University of Texas Southwestern Medical Center,5323 Harry Hines Boulevard, Dallas, TX 75390; hLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Building 4, Room 229, 4 Center Drive, Bethesda, MD 20892; iLaboratory of DNA Replication, TheRockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10021; jDepartment of Pathology, HarvardMedical School, NRB, 77 Avenue Louis Pasteur, Boston, MA 02115; kDepartment of Cell Biology, The Scripps Research Institute, 10550North Torrey Pines Road, La Jolla, CA 92037; lDepartment of Biochemistry and Biophysics, University of California School of Medicine,Howard Hughes Medical Institute, Box 0448, HSE 1001, San Francisco, CA 94143; and mDepartment of Biochemistry, StanfordUniversity School of Medicine, Stanford, CA 94305

Contributed by Stephen C. Harrison, May 21, 2004

Smallpox, a devastating infectiousdisease dreaded throughout muchof recorded history, is caused bythe variola virus, a member of

the poxviridae family. In the 20th cen-tury alone, smallpox deaths worldwidenumbered in the millions. In 1980, afteran intensive program of immunizationwith vaccinia virus, a related but rela-tively nonpathogenic virus, the WorldHealth Organization (WHO) declaredthe disease eradicated. By 1983, allknown stocks of variola virus were intwo WHO collaborating centers: theU.S. Centers for Disease Control andPrevention (CDC) in Atlanta and (aftera transfer in 1994) the Russian StateResearch Center of Virology and Bio-technology (the Vektor Institute) inNovosibirsk. The WHO Committee onOrthopoxvirus Infections voted on sev-eral occasions to recommend destruc-tion of the stocks, but each time thedecision was deferred to permit moreresearch on live variola virus. A 1999National Academies report summarizedand assessed scientific needs for livevariola virus (1).

The concern that undeclared stocks ofvariola virus might exist and that theymight be used as a bioterrorist weapon(2) was heightened in late 2001 by thedeliberate release of Bacillus anthracis,the agent of anthrax, in the weeks afterthe September 11, 2001, attacks. Thatconcern prompted a voluntary national-preparedness effort to vaccinate health-care workers, first responders, andmembers of the military against small-pox. However, given the substantial sideeffects, the risks associated with thesmallpox vaccine, and the absence ofinformation about an imminent bioter-rorist attack, vaccination was not ac-cepted by all members of those groups,nor was it recommended for the generalpublic by the government (3).

Whatever the likelihood of covertlyheld variola virus stocks, an intentionalrelease of the virus would pose a serioushealth threat and would probably pro-voke a global health crisis. The lethalityof the disease (up to 40%) and its easeof transmissibility place variola virus atthe top of CDC’s list of high-threat(Category A) agents. For that reason,the federal government rapidly in-creased its support of research relatedto the discovery of antiviral drugsagainst smallpox. In addition to provid-ing potential therapy for infected peo-ple, the availability of antiviral drugscould decrease the risks associated withthe smallpox vaccine by providing atreatment for vaccine-associated compli-cations. Ultimately, the development ofantiviral drugs against smallpox coulddeter rogue states and terrorists fromreleasing variola virus because its impactwould be diminished. Moreover, thesame new therapies are likely to be use-ful in other poxvirus diseases, such asmonkeypox.

Scientific OpportunitiesIntroduction to Poxviruses.n The poxvirusfamily. Poxviruses are the largest knownanimal viruses, with �200 distinct genes(5). They are DNA viruses that replicateentirely in the cytoplasm. Thus, a subsetof their gene products carries out thefunctions that are essential for the vi-ruses to be independent of the host-cellnucleus. The other viral gene productsuse or modulate an astonishingly widearray of host-cell and immune-systemprocesses.

Poxviruses infect most vertebrates andinvertebrates, causing a variety of dis-eases of veterinary and medical impor-tance. The one large family (Poxviridae)has two main subfamilies, the chor-dopoxvirinae, which infect vertebrates,and the entomopoxvirinae, which infectinsects (Table 1). Each of the chor-

dopoxviruses has a restricted and spe-cific host array (Table 2). Humans arethe sole hosts of two poxviruses, variolavirus (smallpox virus) and molluscumcontagiosum virus, although many mem-bers of Orthopoxvirus, Parapoxvirus, andYatapoxvirus can infect both animals andhumans (Table 2, zoonotic viruses).Vaccinia virus is the virus used in thevariola virus vaccine, and it is widelyused as a model poxvirus in the labora-tory. Its origins are obscure.

The genomes of 30 poxviruses havebeen completely sequenced, includingseveral strains of variola and vacciniaviruses. At least two variants of variolavirus are known, and they cause twoforms of smallpox: variola major, witha case fatality rate of 30–40%, andvariola minor, with a much reducedfatality rate of �1%. At the genomelevel, the two variants are very similar.They differ in �2% of the roughly185,000 unique DNA nucleotides; es-sentially all of the encoded proteinsare nearly identical. These comparisonssuggest that the high fatality rate asso-ciated with variola major may be at-tributable to a small number of geneticdeterminants, and they point to ourignorance of the causes of poxviruspathogenesis in general and of variolavirus pathogenesis in particular. Eradi-

Abbreviations: WHO, World Health Organization; CDC, U.S.Centers for Disease Control and Prevention; IMV, intracel-lular mature vaccinia virion; EEV, extracellular envelopedvirion; ITR, inverted terminal repetition; IEV, intracellularenveloped virion; CEV, cell-associated virion; UDG, uracilDNA glycosylase; IV, immature virion; NIH, National Insti-tutes of Health; SARS, severe acute respiratory syndrome;FDA, Food and Drug Administration; DHHS, Department ofHealth and Human Services; NIAID, National Institute ofAllergy and Infectious Diseases; BSL, biosafety level.

bTo whom correspondence should be addressed. E-mail:[email protected].

nAdapted with permission from ref. 4 (Copyright 2003, ASMPress).

© 2004 by The National Academy of Sciences of the USA

11178–11192 � PNAS � August 3, 2004 � vol. 101 � no. 31 www.pnas.org�cgi�doi�10.1073�pnas.0403600101

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cation of smallpox occurred at a timeof limited knowledge of the molecularand cellular nature of the relevant hostdefense. Thus, during the times when itwas possible to study human infections,the molecular tools for dissecting thehost response were lacking.

Vaccinia virus has become the modelof choice, and our knowledge of poxvi-

rus biology is derived largely from stud-ies of this virus. The tools available forits study now include large collections oftemperature-sensitive mutant strains aswell as recombinant strains with specificgenes under inducible regulation. Sev-eral of the known gene products, suchas its enzymes, can be expressed andstudied in vitro, and new tools of cell

biology are in place to dissect the virus–cell interactions.Poxvirus replication cycle. Poxviruses have acomplex structure. Fig. 1A shows anelectron micrograph of a cross sectionof the infectious intracellular maturevaccinia virion (IMV) and a schematicsummary of the virion. The nature of themembrane envelope surrounding the IMV

Table 1. The poxvirus family: Poxviridae

Subfamily Genus Type species

Chordopoxvirinae Orthopoxvirus Vaccinia virus(vertebrate hosts) Parapoxvirus Orf virus

Avipoxvirus Fowlpox virusCapripoxvirus Sheeppox virusLeporipoxvirus Myxoma virusSuipoxvirus Swinepox virusMolluscipoxvirus Molluscum contagiosum virusYatapoxvirus Yaba monkey tumor virus

Entomopoxvirinae Entomopoxvirus A Melolontha melolontha entomopoxvirus (MMEV)(insect hosts) Entomopoxvirus B Amsacta moorei entomopoxvirus (AMEV)

Entomopoxvirus C Chironomus luridus entomopoxvirus (CLEV)

Table 2. Hosts of and infections caused by chordopoxvirinae

VirusAnimal

reservoir Geographic distribution Transmission to other hosts

Genus MolluscipoxvirusMolluscum contagiosum Humans Worldwide No transmission to other hosts

Genus OrthopoxvirusZoonotic viruses

Monkeypox virus* Squirrels West and Central Africa Monkeys, humansVaccinia virus Unknown Worldwide Humans, buffaloes, rabbits, cowsBuffalopox virus† Unknown Asia Buffaloes, humansCamelpox virus Camels Africa, Asia CamelsCowpox virus‡ Rodents Europe, Asia Cats, cows, zoo animals, humansElephantpox virus Unknown Asia Elephants, humans

Nonzoonotic virusesVariola virus Humans Previously worldwide Only humansVolepox virus Voles Western United States NoneEctromelia virus Rodents Europe NoneRaccoonpox virus Raccoons Eastern United States NoneSkunkpox virus Skunks Eastern United States NoneUasin Gishu disease virus Unknown East Africa HorsesTaterapox virus Gerbils West Africa None

Genus ParapoxvirusZoonotic viruses

Bovine papular stomatitis virus Cattle Worldwide HumansOrf virus Sheep Worldwide Ruminants, humansPseudocowpox virus (milker’s nodules) Cattle Worldwide HumansSealpox virus Seals Worldwide Humans

Nonzoonotic virusesAuzduk disease virus Camels Africa, Asia NoneParapoxvirus of red deer in New Zealand Deer New Zealand NoneChamois contagious ecthyma virus Chamois — —

Genus YatapoxvirusTanapox virus Rodents East and Central Africa Monkeys, humansYaba monkey tumor virus Monkeys West Africa Humans

—, no data available. Table data are taken from Krauss et al. (6).*Can be spread by exotic pets (brought to the U.S. via this route in 2003).†Closely related to vaccinia virus.‡Probably identical to elephantpox virus.

Harrison et al. PNAS � August 3, 2004 � vol. 101 � no. 31 � 11179

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particle remains controversial. Some arguethat there are two closely opposed lipidbilayer membranes; others argue thatthere is one. (The answer has importantimplications for viral entry and matura-tion, as will be discussed below). ThisIMV envelope forms the outer boundaryof a 300-Å-thick surface layer that sur-rounds the inner core, which often ap-pears dumbbell-shaped. The outer row ofreproducibly observed knobs on the corewall has been termed the palisade. Thecore contains the double-stranded viralDNA genome and virion enzymes, includ-ing DNA-dependent RNA polymeraseand RNA-processing enzymes. Pox virions

contain no obvious helical or icosahedralnucleocapsid. A second infectious particle,the extracellular enveloped virion (EEV),contains an additional lipid bilayer mem-brane that is wrapped around the entireIMV particle.

The genomes of poxviruses are dou-ble-stranded DNA molecules. Fig. 1Bsummarizes information obtained fromthe completely sequenced Copenhagenand WR strains of vaccinia virus. The191-kbp genome is a double-strandedDNA molecule whose ends are co-valently connected by single-strandedhairpin loops of 101 nucleotides. Thesequences that form the hairpins are

AT-rich and lie at the ends of 12-kbpinverted terminal repetition (ITR) ele-ments that contain short direct repeatsand several ORFs. The genome se-quence reveals some 185 putative pro-tein-coding sequences and provides adetailed genetic map.

The basis for naming the viral pro-teins is illustrated by the coding se-quences of HindIII fragment D, whichare shown in the expanded view in Fig.1B. The ORFs are depicted by the ar-rows indicating their orientation in thegenome, and each is named with theletter of the HindIII fragment in whichthe first ATG of the reading frame lies,

Fig. 1. Vaccinia virus, a representative poxvirus: virion structure (A) and genome organization with an expanded view of the HindIII D restriction enzymefragment (B). The presence of an inner membrane in the IMV form of the virion shown in A is controversial. See Poxvirus replication cycle for a detaileddescription. [Reproduced with permission from ref. 4 (Copyright 2003, ASM Press).]

11180 � www.pnas.org�cgi�doi�10.1073�pnas.0403600101 Harrison et al.

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followed by a number indicating its or-der and its orientation, either left (L) orright (R). The identities of the encodedproteins, where known, also are listed.

As illustrated in Fig. 1B, the vacciniacoding sequences are densely packed,and the template for RNA synthesis maybe present in either strand. The se-quences encoding structural proteinsand essential enzymes are clustered inroughly the central 120 kb, whereasgenes encoding virulence proteins, host-range proteins, or immunomodulatorsare found predominantly near the ends.This arrangement is also true for allother poxviruses.

Proteins that fulfill different functionsare synthesized during different phasesof the infectious cycle. For example, themRNA capping enzyme, uracil DNAglycosylase (UDG), and most RNApolymerase subunits are products ofearly genes, whereas the genes encodingstructural proteins are expressed onlyduring the late phase of infection. Inaddition, a small but distinct class ofproteins including the late transcriptionfactors is encoded by intermediategenes.

Even though they are DNA viruses,poxviruses replicate in the cytoplasm.Accordingly, they are only minimallydependent on the host cell for DNA andRNA replication, and they encode theirown proteins for these processes. A sim-plified version of the single-cell repro-ductive cycle of vaccinia virus is illus-trated in Fig. 2. EEV entry is illustratedin Fig. 2, step 1. The mechanisms bywhich either the IMV or the EEV infec-tious forms of vaccinia virus attach toand enter susceptible host cells are notwell understood and will be constrainedby the number of membranes envelopingthe virus. After fusion of the viral andcellular membranes, primary uncoatingtakes place, and the viral core is re-leased into the cytoplasm. All later stepsin the infectious cycle take place in thiscellular compartment. The core con-tains, in addition to the viral genome,the viral DNA-dependent RNA poly-merase, the ‘‘initiation’’ proteins necessaryfor specific recognition of the promotersof viral early genes, and several RNA-processing enzymes that modify viraltranscripts. On release into the host-cellcytoplasm, the core synthesizes viralearly mRNAs (Fig. 2, step 2), which ex-hibit the features typical of cellularmRNAs and are translated by the cellu-lar protein-synthesizing machinery (Fig.2, step 3). Approximately half of theviral genes are expressed during thisearly phase of infection.

Some early proteins are secreted fromthe cell (Fig. 2, step 4) and have se-quence similarity to cellular growth fac-

tors, which can induce proliferation ofneighboring host cells, or are proteinsthat counteract host immune defensemechanisms. The synthesis of early pro-teins also induces a second uncoatingreaction in which the core wall opensand a nucleoprotein complex containingthe genome is released from the core(Fig. 2, step 5). This core disassemblyleads to cessation of viral early geneexpression. Other early proteins catalyzethe replication of the viral DNA ge-nome (Fig. 2, step 6). Newly synthesizedviral DNA molecules can serve as tem-plates for additional cycles of genomereplication (Fig. 2, step 7), and they arethe templates for transcription of viralintermediate-phase genes (Fig. 2, step8). The activation of transcription ofintermediate genes also requires specificviral proteins (the products of earlygenes) that confer specificity for inter-mediate promoters on the viral RNApolymerase, as well as a host-cell pro-tein (Vitf2) that relocates from theinfected cell nucleus to the cytoplasm.The proteins encoded by intermediatemRNAs (Fig. 2, step 9) include thosenecessary for transcription of late-phasegenes (Fig. 2, step 10). The latter genesencode the proteins from which virionsare built as well as the virion enzymesand other essential proteins, such as theearly initiation proteins, that must beincorporated into virus particles duringassembly. Once these proteins are

synthesized by the cellular translationmachinery (Fig. 2, step 11), the assem-bly of progeny virus particles begins.The viral membrane proteins are ungly-cosylated, and the role of cellular mem-branes in early stages of assembly iscontroversial (Fig. 2, step 12).

The initial assembly reactions result information of the immature virion (Fig.2, step 13), which is a spherical particledelimited by a membrane that may beacquired from an early compartment ofthe cellular secretory pathway. This vi-rus particle matures into the brick-shaped IMV (Fig. 2, step 14), which isreleased only on cell lysis (Fig. 2, step15). However, the particle can acquire asecond, double membrane from a trans-Golgi or early endosomal compartmentto form the intracellular envelopedvirion (IEV) (Fig. 2, step 16). The IEVsmove to the cell surface on microtubuleswhere fusion with the plasma membraneforms cell-associated virions (CEV)(Fig. 2, step 17). These CEV induce anactin polymerization that promotes adirect transfer to surrounding cells (Fig.2, step 18); they can also dissociate fromthe membrane as EEV.

Although studies of viral entry andmorphogenesis have revealed strikingmechanisms by which the viral proteinsinteract with the actin and microtubulecytoskeleton, they also have left us withseveral unresolved puzzles in membranebiogenesis. In addition, there is an ex-

Fig. 2. The single-cell reproductive cycle of vaccinia virus. The entry and replication of an EEV areillustrated. RNA molecules are green. See Poxvirus replication cycle for a detailed description of eachillustrated step. [Reproduced with permission from ref. 4 (Copyright 2003, ASM Press).]


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tensive molecular dialogue between theviral gene products and the vertebratehost’s innate and adaptive immune sys-tems, as illustrated by the known targetsof many of the virus-encoded immunemodulators and regulators of apoptosis(Fig. 3). These include secreted ‘‘viro-kines’’ that mimic host cytokines, se-creted ‘‘viroceptors’’ that mimic hostcytokine receptors, and intracellularproteins that interfere with apoptosisand signaling pathways.

In short, it is clear that poxvirusesconstitute an underexploited tool forprobing fundamental processes in bothcell biology and immunology. By takingadvantage of this tool, we will certainlyincrease our understanding of importantbiological mechanisms; we are also verylikely to discover unexpected ways tocontrol poxvirus infections.

Routes to New Antiviral TherapeuticAgents: Poxvirus Enzymes as CandidateDrug Targets. Essential viral enzymeshave frequently proved to be good tar-gets for antiviral drugs (for example,herpesvirus thymidine kinase, HIV re-verse transcriptase and protease, andinfluenza neuraminidase). Initial effortsto identify inhibitors should thereforefocus on enzymes that have essentialroles in poxvirus replication and forwhich atomic structures have beendetermined or can be determined

promptly. These criteria narrow the fieldof candidate targets considerably. So far,the atomic structures of five poxvirus pro-teins have been determined: topoisomer-ase I, cap-specific 2�O-methyltransferase�poly(A) polymerase stimulatory subunit,complement-control protein, CrmA im-mune modulator, and an inhibitor of theRNA-dependent protein kinase. A pox-virus structural-biology initiative wouldbe a valuable component of an orga-nized effort to discover inhibitors of vi-ral enzymes. The enzymes listed inTable 3 have at least some of the de-sired characteristics.

Drugs that target topoisomerases andcause them to damage DNA are amongthe most successful therapeutics devel-oped for both infectious disease (quino-lones) and cancer (etoposide, adriamycin,and camptothecins). Indeed, drug screen-ing ‘‘in silico’’ (that is, by computermodeling) is already under way for pox-virus topoisomerase I, a 314-aa mono-mer comprising two domains joined by aflexible molecular hinge. A recent studyindicates that the enzyme is not abso-lutely essential but has an importantrole in enhancing early transcription inthe virus core (13).

H1 protein phosphatase, a dual-speci-ficity cysteine phosphatase encapsidatedin the virus particle, is required in orderfor the encapsidated viral genome to betranscribed by the viral transcriptase.

Dual-specificity phosphatases (DSPs)participate in a number of cellular sig-naling pathways. Their activities are rel-evant to various disease states, andthere has been substantial work on theirpharmacology. The DSP domain of H1is small (171 aa), however, and lacksunique structural features. There arealso highly conserved ‘‘vaccinia H1-like’’homologues in humans. Thus, a bettercandidate for an antiviral might be theF10 dual-specificity protein kinase,which also is critical for the earlieststeps of virus morphogenesis. It has nosequence similarity to known cellularkinases, with the exception of a 110-aaregion of a protein known as human H1protein phosphatase. The F10 proteinsequences in variola and monkeypoxviruses are 98% identical.

The I7 cysteine protease, encapsi-dated in the virus particle, processes themajor virion core proteins at a cleavagemotif, AlaGly1X. A loss in functional-ity of the enzyme leads to arrest ofvirion morphogenesis at a point afterthe formation of spherical immatureparticles. This protein of 432 aa is 99%identical in variola and monkeypox vi-ruses; it appears to have only a verydistant relationship to the cellularSUMO-specific protease Ulp1 and toadenovirus and African swine fevervirus (ASFV) proteases.

The A22 Holliday junction (HJ) re-solvase is a small, 187-aa, metal-depen-dent nuclease. The encapsidated enzymeis synthesized late in infection and par-ticipates in the resolution of concate-meric DNA replication intermediatesbefore genome packaging. A distant rel-ative of the bacterial HJ resolvase RuvCand fungal mitochondrial resolvaseCce1, the nuclease is peculiar to poxvi-ruses and extocarpus siliculosus virus;no human orthologues have been identi-fied. Little is known about possible HJnuclease inhibitors and their potential asdrugs.

Uracil DNA glycosylase (UDG) isa 218-aa protein that is required forDNA replication. The UDG has anessential role in viral DNA replicationthat is independent of its catalytic ac-tivity. However, viruses encoding a cat-alytically inactive UDG are attenuatedin vivo.

The poxvirus capping enzyme is amultisubunit complex responsible foradding a 7-methyl guanosine ‘‘cap’’ tothe 5� end of viral messenger RNA. Al-though they accomplish the same func-tion, there are important biochemicaldifferences between the capping com-plexes of poxviruses and those of theirmammalian hosts.

Fig. 3. Schematic representation of selected poxvirus immunomodulators and regulators of apoptosis.Proteins shown in red represent poxvirus proteins; host proteins are shown in black and gray. Secretedpoxvirus viroceptor proteins (top row) function as soluble or cell surface decoys that bind host-cellcytokines or chemokines in the cell exterior. Poxvirus virokines also are secreted; they function as agonisticor antagonistic ligands for host cellular receptors (middle row). A number of poxvirus proteins functioninside the cell to modulate apoptosis, cytokine processing, and host range (red proteins in cell interior).[Reproduced with permission from ref. 7 (Copyright 2003, Annual Reviews, www.annualreviews.org).]


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The virion encapsidated RNA poly-merase (RNAP) holds great potential asa drug target. This nine-subunit enzyme,packaged in the viral core, transcribesgenes expressed early in replication.Many of the subunits are not found incellular RNA polymerases. RNAP ap-pears to have a unique initiation mecha-nism that requires early transcriptionfactors (ETFs) found only in poxviruses.Attempts to target pox RNAP so farhave taken two approaches. The first isto use small molecules to block pro-moter recognition or ATP hydrolysis byETFs. The second is to devise an‘‘mRNA poison’’: here the virus is pro-vided with a substrate analogue (forexample, adenosine N1 oxide) that isincorporated into the viral mRNA bypoxvirus RNAP, but the mRNA cannotbe translated into viral proteins.

The poxvirus poly(A) polymerase is aheterodimer composed of catalytic andprocessivity subunits. The processivitysubunit, for which the atomic structureis known, also methylates the ribose ofmRNA caps and works as a transcrip-tion elongation factor.

The E9 DNA polymerase, requiredfor DNA replication, acts in concertwith accessory proteins to attain effi-cient processive synthesis. Accessoryproteins include the A20 protein, D4UDG, and possibly others. The D5NTPase and the viral SSB (the I3 protein)also are essential for viral replication. TheNTPase has homology to helicases, al-though it is not yet known to act in thiscapacity. Viral DNA polymerases areestablished drug targets, as exemplifiedby azidothymidine (AZT), which inhibitsthe HIV reverse transcriptase, and acy-clovir, which is efficiently phosphory-lated by the herpes simplex virus viralthymidine kinase, resulting in a triphos-phate that preferentially inhibits viral

DNA polymerase. The DNA polymeraseinhibitor cidofovir inhibits poxvirusDNA polymerases, including that of var-iola virus, and it has been shown tohave antiviral effects in animal models.The drug currently is used to treat mol-luscipoxvirus infection in AIDS patientsand is recommended by CDC for use intreating complications of vaccinationwith existing smallpox vaccines.

Three conserved poxvirus thiol oxi-dases, only distantly related to cellularproteins, are required for formation ofthe disulfide bonds in several viral mem-brane proteins. Deletion or mutation ofany of these proteins prevents virus as-sembly. Thus, each of these redox pro-teins would provide a good target fordeveloping an antiviral drug.

Cell Biology of Poxviruses. Six aspects ofpoxvirus research are particularly ripefor investigation and offer substantialopportunities for discovery. They are: (i)the mechanism of entry into host cells;(ii) the regulation and intracellular orga-nization of DNA replication, DNA tran-scription (RNA synthesis), and translation(protein synthesis); (iii) the viral manipu-lation of host-cell membrane traffic; (iv)the viral subversion and exploitation ofhost-cell signaling pathways; (v) the viralexploitation of motor proteins for move-ment within a cell and for propulsiontoward another cell; and (vi) the mecha-nism of cell-level host-range restriction.This list of topics indicates the wide ar-ray of advanced scientific expertise thatshould be used in future studies of pox-virus biology to increase our under-standing of the infectious process. Fig. 4highlights features of poxvirus cell biol-ogy and some of the unanswered ques-tions for future research.Entry into host cells. Vaccinia virus caninvade and replicate in a wide array of

host-cell lines, but the identity of host-cell receptors and the routes of entryinto the cytosol remain unknown (Fig.4, step 1). There are two infectiousforms of the virus, IMV and EEV, witheither one or two (IMV) or two or three(EEV) lipoprotein bilayers surroundingthe nucleoprotein core. The identity ofputative viral fusion proteins is un-known, but the unusual arrangement ofthe poxvirus envelope may imply that itsfusogens are mechanistically distinctfrom more familiar viral fusion proteins.Identification and characterization ofthe entry, fusion, and uncoating mecha-nisms of poxviruses should thereforebroaden our understanding of the gen-eral mechanism of membrane fusion,which is central to a wide variety of crit-ical processes in cells.Regulation and organization of DNA replica-tion, transcription, and translation. Poxvirusesare unique among animal DNA viruses incarrying out DNA replication and tran-scription in the interphase cytoplasm. Ex-pression of the viral genome thus includesopportunities for types of regulation unfa-miliar elsewhere in animal cell biologyand hence opportunities to identify spe-cific antiviral targets. Only the bare essen-tials of the regulation of poxvirus geneexpression are understood. Promoter se-quences, the virus-encoded RNA poly-merase, and transcription factors havebeen identified (14), but the mecha-nisms of transcription initiation, elonga-tion, and termination are not known inany molecular detail. Data from in vitrostudies indicate a role of host proteinsin transcription, but no supporting invivo data have been reported. It haslong been known that intermediate andlate transcripts have nontemplated 5�poly(A) leader sequences, but their rolehas not been clarified. After virus infec-tion, the turnover of all mRNAs is

Table 3. Potential drug targets: Selected poxvirus enzymes

Drug target Poxvirus enzymes

DNA replication and recombination (Fig. 2, steps 6 and 7)E9 DNA polymerase (target of cidofovir)A20 Polymerase processivity factorA22 Holliday junction resolvaseD4 Uracil DNA glycosylaseD5 Nucleoside triphosphatase

mRNA synthesis and modification (Fig. 2, steps 2, 8, and 10)Nine subunits RNA polymerase (J6, A24, A29, E4, J4, A5, D7, G5.5, and H4)E1 (�J3) Poly(A) polymerase [catalytic (�stimulatory)]D1 (�D12) mRNA capping enzyme [catalytic (�stimulatory)]H6 Topoisomerase IH1 Protein phosphatase (dual specificity)

Protein modification and virus assembly (Fig. 2, step 14)F10 Protein kinase (dual specificity)I7 Cysteine proteaseE10, A2.5, G4 Thiol oxidases


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greatly increased by an unknown mecha-nism. With regard to DNA replication,several essential proteins have beenidentified by genetic screens, but theirfunctions, except for that of the DNApolymerase, are not well defined (5).Other features of poxvirus DNA replica-tion deserve attention. The linear DNAgenome is terminated by hairpin loopsat each end and is thus a covalentlyclosed, single-stranded DNA. How doesreplication begin on this template? Afocused effort on developing an in vitroDNA replication system composed ofpurified poxvirus proteins capable ofreplicating double-stranded DNA tem-plates could provide a particularly fruitful

test bed for the screening of potential an-tiviral drugs. [For a review of other suchsystems, see Bell et al. (15)].

There are cytologic hints that poxvi-ruses establish some type of membrane-enclosed ‘‘virus factory’’ in thecytoplasm where replication takes place(Fig. 4, step 2). What is the nature ofthis putative compartment, and howdoes the virus set it up? Transcriptionand translation of at least some viralproducts are spatially controlled in thehost cell, and some kinds of directedtransport must take place during virus-particle assembly (Fig. 4, step 4). Whatis the molecular basis of these processes,and what roles do host-cell structural

components have in scaffolding or di-recting them?Viral manipulation of host-cell membrane traf-fic. To what extent does the virus exploithost-cell structures for organization of itsassembly process, and to what extent doesit remodel the cell scaffold to fit its needs(16)? Host membrane alterations areclosely coupled to spatial targeting, trans-port, and assembly of virus-particle com-ponents. An early event in morphogenesisis the appearance within virus-factory re-gions of membrane ‘‘crescents,’’ whichmay be closely apposed lipid bilayers de-rived from endoplasmic reticulum mem-brane (Fig. 4, step 3). How do the viralproteins target, recruit, and grossly rear-

Fig. 4. Major questions in the cell biology of poxvirus infection. (A Center) The schematic portrays some events in vaccinia infection and morphogenesis.The specific steps and unanswered questions are numbered and discussed in the text. Stages of virus maturation shown are immature virion (IV),intracellular mature virion (IMV), intracellular enveloped virion (IEV), cell-associated enveloped virion (CEV), and extracellular enveloped virion (EEV).Images surrounding the schematic illustrate some of these events. (Upper Left) Fluorescent micrographs of viral factories, which are detected togetherwith the cell nucleus by the DNA binding dye Hoescht (blue) and, more specifically, by antibodies to the viral protein encoded by the H5 gene (green)[Reproduced with permission from ref. 8 (Copyright 2001, American Society for Cell Biology).] (Lower Left) A thin-section electron micrograph ofmembrane crescents, an early stage in viral assembly. The derivation of the membrane from either the endoplasmic reticulum (ER) or intermediatecompartment (IR) is uncertain, although regularly spaced small spikes can be seen on the outer membrane (arrows). [Reproduced with permission fromref. 9 (Copyright 2002, American Society for Microbiology).] (Lower Near Right) Superimposed frames from a time-lapsed video showing microtubule-based movement of vaccinia virus that appears as red streaks along GFP-labeled microtubule tracks (green). [Reproduced with permission from ref. 10(Copyright 2001, Nature Publishing Group, www.nature.com).] (Lower Far Right) Viral particles (green) remain stably associated with microtubules (red)even after extensive extraction of infected cells. [Reproduced with permission from ref. 11 (Copyright 2002, Society for General Microbiology).] (UpperRight) A fluorescent micrograph of actin tail formation (green) juxtaposed to and triggered by cell-surface associated CEV (red), which function to propelthe virus particle away from the cell and�or into adjacent cells. 4�,6-Diamidino-2-phenylindole (DAPI) staining (blue) reveals a large viral factory adjacentto the cell nucleus (Photograph courtesy of Tim Newsome and Michael Way). (B) Thin-section electron micrographs of sequential stages of viral maturation.[Reproduced with permission from ref. 12 (Copyright 2001, American Society for Microbiology).]


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range the host membranes to form theseunusual structures, and how are viral ge-nomes delivered to and inserted intothem? Is there any relationship of crescentformation to autophagy? At later stages,the viral particles undergo a second layerof membrane wrapping, this time recruit-ing trans-Golgi or endosomal membranes(Fig. 4, step 5). Analysis of mutants andrecombinant strains has enabled identifi-cation of viral proteins that are involvedin many of these membrane manipula-tions, creating a diverse molecular toolkit,both for exploring cell membranes andorganelles and for providing a diverse ar-ray of targets for inhibitors.Viral exploitation of host-cell signaling path-ways. Before the assembled particle be-comes infectious, an essential virally en-coded protease must cleave severalproteins in a process that is coupled tocondensation of the core. A virus-specificgroup of thiol transferase enzymes alsocatalyzes formation of disulfide bonds onthe intracytoplasmic tails of virus mem-brane, enabling this oxidation to occur inan apparently reducing environment (17).The initial, immature particle, the imma-ture virion (IV), is noninfectious, but thefinal result of the maturation steps, theIMV, is both highly infectious and stable.How does morphogenesis occur, and whatmolecular and mechanical events are in-volved? How is protein processing bycleavage and disulfide formation linked tothe morphologic alterations? What keydifferences determine the infectivity ofthe IMV as opposed to the IV?Viral exploitation of motor proteins. The as-sembled particles use microtubule mo-tors to get to the perinuclear region forenvelopment in the Golgi membrane(Fig. 4, step 4) and subsequently movealong microtubules to the cell surface(Fig. 4, step 6) (18). Moreover, buddedextracellular virions still attached to thecell membrane direct assembly of intra-cellular actin bundles at positions justbeneath their membrane attachmentpoint, driving formation of a membranestalk that bears the virion (CEV) at itstip (Fig. 4, step 7). How do viral pro-teins establish these assemblies? Whichproteins are essential for spread of theinfection in a tissue?Mechanism of host-range restriction in vitroand in vivo. There are a number of mo-lecularly defined mutant vaccinia virusstrains that replicate in some mamma-lian lines but not in others. Thosestrains provide an opportunity to definethe role of host-cell factors in viral rep-lication and morphogenesis. Thesemutant viruses also may facilitate thedevelopment of safer vaccines.

Understanding the complex molecularinteractions that occur between the rep-

licating virus and the host cell shouldreveal novel targets for antiviral therapy.

Research Needs in Pathogenesis and HostResponse. Animal models. Because of thehost-range specificity and containmentrequirements of variola virus, closelyrelated orthopoxviruses that replicate inrodents (such as cowpox virus, ectrome-lia virus, and vaccinia virus, includingthe rabbitpox strain) are usually used forstudies of viral pathogenesis. Monkey-pox in the cynomolgus monkey is thebest nonhuman-primate model, althoughlarge doses of virus are required. Mon-keypox virus is a select agent (a hazard-ous biological agent subject to safetyregulations), and Biosafety Laboratory 3facilities are required, but primate mod-els are essential for testing vaccines andtherapeutics at late stages in their devel-opment. More accessible and less expen-sive animal models will be critical forrapid work at earlier stages, and devel-opment of realistic animal models fororthopoxvirus infection and disease(such as models for which the innocu-lum required for infection is not heroicand for which the endpoint is not neces-sarily death) should be emphasized. Re-search across the entire spectrum ofpoxvirus genera should be strongly en-couraged, because the history of virol-ogy has shown that key insights into onegroup of viruses often come from thestudy of an apparently divergent group.Proteomics. There is a clear need for acomplete analysis of the structural andnonstructural proteins of variola virusand related orthopoxviruses supportedby the requisite bioinformatics tools.Neither infectious variola virus nor in-tact variola virus DNA can be trans-ferred from the two authorized smallpoxfacilities, but WHO regulations allowother laboratories to work with DNArepresenting up to 20% of the variolavirus genome in nonpoxvirus vectors. Acoordinated international effort to studyvariola virus proteins therefore will benecessary if we are to acquire a properunderstanding of the viral proteome.Viral and cellular determinants of host re-sponse. As is the case with many otherviruses, little is known with certaintyabout immune characteristics that corre-late with protective immunity againstvariola viruses or other poxviruses. Con-sequently, all aspects of both the innateand acquired immune response must bestudied. It is important to define theinnate immune response to a poxvirusinfection, including cytokine productionand regulation of cellular receptors in-volved in activation of elements of theinnate immune system. The capabilitiesof modern DNA-array technologyshould be brought to bear on this analy-

sis, with a focus on primate models (7).Fortunately, the available human arraysappear to be useable for nonhuman-primate samples as well.

If an animal model (such as monkey-pox in cynomolgus monkeys) is deemedan adequate surrogate for variola virus,it will be of the utmost importance totrack the various immune characteristicsknown to correlate with B cell and Tcell activation and with emergence ofmemory B and T cells. That goal willrequire establishment (in the BL4 facil-ity for variola virus and in appropriatecontainment facilities for other poxvi-ruses) of the appropriate cell-sortinginstruments, allowing researchers toreadily obtain the relevant leukocytesubsets from infected animals (B cells,T cells, macrophages, dendritic cells,and so forth). Similarly, obtaining puri-fied leukocyte subsets in this way will beessential for evaluating cytokine produc-tion. Wherever a mouse model can beused, advantage can be taken of theoutstanding sets of reagents that areavailable (antibodies, nucleic acids, andknockout animals). Those experimentsshould be started now.Immunotherapeutics. Protective human orhumanized monoclonal antibodies tovariola virus for use in immunotherapyshould be developed with modern tech-nologies. These antibodies would be di-rected to the viral surface proteins andpossibly to secreted products. Many ofthe antibodies would crossreact withother orthopoxviruses and could betested in animal models. The perceivedimportance of a ‘‘cytokine storm’’ (inwhich the production and release ofinflammatory cytokines and stress medi-ators overwhelms the host) as a majordeterminant of the pathologic conditionoffers further opportunities for immuneintervention (for example, with tumornecrosis factor antagonists).Safer vaccines. Further knowledge of or-thopoxvirus proteins, the mechanisms ofinfection, and the correlates of immu-nity may enable the design of safersmallpox vaccines. Possible approachesinclude the development of attenuatedstrains of vaccinia virus, recombinantproteins, and vectors. Any antipoxvirustherapeutics developed will make it pos-sible to treat the rare complications ofvaccination, making vaccinations evensafer.

Discussion. Naturally occurring variolavirus has been eradicated from theplanet. Indeed, the last reported case ofnatural smallpox occurred in Somalia in1977, long before recent advances inmolecular biology, cell biology, and im-munology would have allowed research-ers to study the variola virus and the


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pathogenesis of human infection in finedetail. Studies on the closely relatedvaccinia virus have provided a relativelythorough outline of poxvirus replicationin mammalian cells, but a great dealremains to be understood and little isknown about how the human immunesystem responds to variola virus. Giventhe virulence of this virus (up to 40%mortality) and its ability to spread in apopulation, the consequences of an in-tentional release of variola virus couldbe devastating. Official stocks of thevirus are closely held, but it is notknown whether undeclared stocks exist,so it is difficult to assess the current de-gree of risk. Safer vaccines and therapeu-tics that can mitigate the consequences ofinfection would together provide a strongdeterrent to any intentional release. Effec-tive development of such deterrents willbenefit greatly from a deeper understand-ing of the biology of poxviruses and ofhow they interact with their hosts. Thereis an enormous range of opportunities:because variola is a large virus, it requiresmany specific viral-encoded proteins toinfect humans, each of which represents apotential target for an antiviral drug.

We recommend a research-and-devel-opment response at three levels. First,we propose an immediate focus on cur-rently identifiable therapeutic targets,particularly essential poxvirus-encodedenzymes, through a combination of aca-demic and industrial research. The phar-maceutical industry has enormousexperience in the discovery, design anddevelopment of enzyme inhibitors, andeach of the enzymes listed in Table 3 isa potential antiviral target. We recom-mend that the National Institutes ofHealth (NIH) use the mechanisms out-lined in Incentives, Logistics, and Policiesbelow (see especially Recommendation1) to initiate, both in pharmaceuticaland biotechnology companies and insome collaborating academic laborato-ries, immediate work on each of theseenzymes. Second, we suggest that rapid,short-term progress toward deeper un-derstanding of poxvirus biology will re-sult from active collaborations betweengovernment or academic laboratoriesalready engaged in poxvirus researchand others with specific biologic or tech-nologic expertise. As examples of suchcollaborations, we recommend NIH-sponsored initiatives in the structuralbiology of poxvirus proteins, in the cellbiology of poxvirus infection, and in theinteraction between poxviruses and thehost immune system. Third, we observethat longer-term progress will requirerecruitment and training of a new co-hort of outstanding young investigators,and we suggest that the initiatives justlisted be designed with this long-term

goal in mind. In the second part of thisreport, Incentives, Logistics, and Policies,we consider the organizational require-ments for achieving each of those objec-tives and propose specific ways to meetthem.

There is a pressing need for new anti-viral drugs and new vaccines, whether tocounter potential bioterrorist threats, totreat HIV, or to deal with emergingpathogens like severe acute respiratorysyndrome (SARS) coronavirus. Collabo-rations that bring poxvirologists togetherwith outstanding scientists in otherfields of biology are not only likely toaccelerate the search for ways to controlinfection by variola virus but also toproduce discoveries relevant to diversepathogens, including other DNA virusesand intracellular bacteria. Such collabora-tions will also expand our understandingof the fundamental cellular processes thatpoxviruses exploit.

In the broadest sense, directing theattention of a larger number of out-standing scientists with expertise in cellbiology, structural biology, computa-tional biology, and chemical biology tothe complexities of viral pathogenesiscan be a fruitful effort for increasingboth national and international security.In the long run, a well conceived, con-certed program to discover treatmentsfor smallpox (should it reemerge) willprovide models for how to motivate in-terdisciplinary groups to solve other ma-jor problems in public health. It willlikewise demonstrate how to create ef-fective incentives for participation of thebiotechnology and pharmaceutical in-dustries in the production of new typesof protective agents against viruses andother threat agents, whether these areintroduced intentionally or through nat-ural processes.

Incentives, Logistics, and PoliciesIt is not known whether there are stocksof variola virus other than those sanc-tioned by the WHO or whether small-pox will someday become a weaponused by terrorists or rogue states. If thelatter were to occur, the reemergence ofsmallpox would create a major interna-tional health crisis. To counter the po-tential use of variola virus as a weaponand to protect the public in the event ofan intentional release, a concerted inter-national effort is needed to tackle thescientific challenges described in thefirst part of this report and to translatethe results of that research into newtherapeutic candidates.

It is important to recognize that theeradication of naturally occurring small-pox was achieved through a global ef-fort. Therefore, although many of therecommendations related to those issues

are formulated here in the context ofU.S. institutions (for example, NIH)the committee urges that its proposalsbe taken in an international context andthat the initiatives recruit broad inter-national participation.

Engaging the Pharmaceutical and Biotech-nology Industries. The importance of indus-trial participation at early stages in the discov-ery of smallpox antivirals. If the discoveryand development of antiviral therapeu-tics to treat poxvirus infections is to bea high priority, it is essential that phar-maceutical and biotechnology companiesbe engaged from the outset. Academicresearchers eventually may acquire theresources, equipment, compound librar-ies, and experience required to partici-pate successfully in drug discovery, butthat process will certainly take years.Meanwhile, there are many pharma-ceutical and biotechnology companiesalready poised to discover poxvirus-specific antiviral drugs, beginning withthe candidate targets listed in Table 3.These companies currently have theequipment and compound libraries re-quired to screen more than a millioncompounds per month against any drugtarget peculiar to poxviruses. Moreover,these for-profit companies have the ex-pertise needed to assess the attributesor deficits of compounds emerging atearly stages from high-throughput drugscreens; to synthesize the hundreds ofanalogues of early drug candidates re-quired to move such compounds fromthe early ‘‘hit’’ stage into bona fide drugleads; to perform the pharmacologicand toxicologic assessments essential forthe generation of substantive, preclinicaldrug leads; to perform the detailed,regulated preclinical studies needed forsubmission of an investigational newdrug (IND) application to the Food andDrug Administration (FDA); and toperform clinical trials at the safety andefficacy stages of drug development.

The success of both traditional phar-maceutical companies and biotechnologycompanies in discovering, developing,and marketing novel therapeutics spe-cific for the treatment of HIV demon-strates the power of these organizationsfor producing clinically valuable antiviraltherapeutics. Poxviruses offer a farbroader spectrum of therapeutic targetsthan does HIV, and it is reasonable toexpect that finding poxvirus-specificdrugs will be less demanding than thechallenges presented by HIV. We there-fore believe that, with the engagementof appropriate pharmaceutical and bio-technology groups, clinically valuableproducts will emerge.


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A program for engaging the pharmaceuticalindustry. Engaging pharmaceutical andbiotechnology companies in a poxvirusdrug-discovery effort will require signifi-cant changes in the ways that NIH,CDC, and other federal agencies havetraditionally interacted with industry.First, because the government is likelyto be the sole purchaser of any prod-ucts, their development will require in-centives and resources at critical stages.Second, the way in which programs andproposals are evaluated and reviewedwill require advisory structures and pro-cedures quite different from the peer-review system that works well for aca-demic research awards.

The committee suggests the followingoutlines of a program to meet the chal-lenges of industry incentives. First, se-nior government health officials shouldmake a direct and open invitation to thehighest levels of pharmaceutical andbiotechnology management. A widelyannounced, high-profile program wouldallow companies to be seen as contribut-ing to an important public-health re-sponse. Second, NIH should providebroad support for the early stages ofdrug discovery, beginning with currentlyidentifiable targets and continuing asbasic research uncovers new targets andnovel strategies. Third, several awards(perhaps five or six in total) should bemade to companies that have demon-strated their ability to deliver the mostattractive preclinical candidate drugs,derived from work in the previous stage.These awards would support acceleratedpreclinical drug development, including‘‘good manufacturing process’’ produc-tion, full evaluation of drug metabolismand pharmacokinetics, toxicology andanimal efficacy tests, and appropriateregulatory compliance leading to an in-vestigational new drug submission to theFDA.

The fourth step of the proposed anti-poxvirus drug development programincludes clinical studies and preparationof a new drug application (NDA) forapproval by the FDA. This will be themost expensive phase of the process,and it is particularly for this step thatnew government�industry relationshipswill need to emerge. One potentialstrategy, which is incorporated in thepending BioShield legislation (see be-low), involves the concept of a guaran-teed market, with prenegotiated pricescontingent on successful product devel-opment (as defined by FDA licensing)within a fixed time frame. Key decisionswill need to be made about the negotia-tion of a guaranteed market and priceand about the timing and amount ofgovernment support during the develop-ment process. A possible drawback of

this process is that it tends to rewardthe first company to bring forward adrug rather than the one that producesthe best drug.

An alternative strategy for step fourwould be to create a ‘‘public pharma-ceutical company,’’ either specifically fordevelopment of smallpox antivirals ormore broadly for other bioweaponscountermeasures. Such a course would,however, require the duplication oftalent and resources already availablewithin the pharmaceutical industry. Thelong timescale for drug developmentalso might be inappropriate for a singlemission-oriented facility.

It would be desirable to have the par-ticipation of pharmaceutical companiesof various sizes in the discovery and de-velopment process. The committeefound, in discussions with individualsacquainted with both large pharmaceuti-cal companies and smaller biotechnol-ogy firms, that engaging companies inthese two categories might require dif-ferent approaches. For example, thecontract program outlined as steps twoand three in the preceding paragraphwould probably work better for smallercompanies, whereas larger firms mightrespond better to a single contract cov-ering both these steps, specifying appro-priate milestones for continuation.

The government also might considerassembling a consortium of pharmaceu-tical and biotechnology companies toshare knowledge as discovery proceeds.In this setting, the exchange of informa-tion would reduce individual opportu-nity costs. Moreover, if one companywere to discover a particularly favorablesynthetic scaffold or series of com-pounds that inhibit a specific target, itwould make sense for the entire com-munity to focus on it. Alternatively, itmight be appropriate to distribute workon different targets among differentparticipating companies. Because thegovernment is not the customary marketfor pharmaceutical companies, and be-cause knowledge-sharing might lead toadvances in understanding of antiviralsin general, pharmaceutical companiesmight be more willing than usual to ex-change information. The issues of anti-trust regulation and intellectual propertyprotection raised by such information-sharing would need to be considered,but if the program were deemed a suffi-ciently critical national priority, theyshould not be insurmountable.

The committee believes that in settingup and implementing a program such asthe one outlined above, it is especiallyimportant that wise decisions be madeby using the best scientific judgment andastute management considerations.Moreover, the ways in which programs

are implemented will need to respond tothe uncertainties and surprises of discov-ery and development. The concept oflarge, long-term contractual relation-ships with for-profit companies is rela-tively new to NIH and to the Depart-ment of Health and Human Services(DHHS). However, the National Insti-tute of Allergy and Infectious Diseases(NIAID) has had some experience inthis type of decision-making, boththrough its AIDS vaccine research effortand through the creation of the intramu-ral Vaccine Research Center.

The high-level advisory and oversightpanel described below in the section Im-plementation should have a major role inguiding the interaction of NIH and in-dustry. In addition, the leadership ofNIH and DHHS should examine howother branches of the federal govern-ment, such as the Department of De-fense, interact with the private sector, asit develops its own model for such col-laboration.

NIH will be defining goals and steer-ing research. It therefore will be essen-tial to develop effective modalities ofinternal and external review so that NIHcan respond rapidly to proposals underthe multistep program outlined aboveand so that it can have effective inputfrom individuals with experience andgood judgment in practical drug discov-ery and development. The mechanismsof peer review used by NIH to set prior-ities for support of academic science willbe inadequate for evaluating programsthat require large, long-term funding ofprivate-sector research and develop-ment, both because conventionalacademic review groups tend to be rela-tively conservative and because thecontract mechanism requires differentqualities than a grant.

The committee notes that AnthonyFauci, director of NIAID, already hasbroached the subject of antiviralsagainst smallpox with the heads of sev-eral leading pharmaceutical and biotech-nology firms, initiating what we definehere as step one.

As a rough measure of investmentcosts, the committee offers the followingoutline. Recent research, based on asurvey of a large number of companies,finds that development of a single newdrug costs �$800 million (year 2000dollars) in out-of-pocket expenses, capi-talized to the point of marketing ap-proval (19). This estimate, which may bethe high end of a spectrum of possiblevalues, includes the costs of failed candi-dates at various stages in the develop-ment process. Clinical trials will be lessexpensive in the case of smallpox antivi-rals, because efficacy tests will involveanimal rather than human subjects, but


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the cost savings might be offset by therequirements of a shorter-than-averagetimeline. The committee therefore choseto use the estimate $800 million for theoverall cost of developing a single drug.Procurement, stockpiling, and renewalcosts are, of course, additional. (Re-newal costs refer to expenses requiredto ascertain potency of stockpiled mate-rials and to replace them as needed.) Itremains to be determined whether con-tract support for all or a substantialfraction of the $800 million would offsetopportunity costs sufficiently to inducecompanies to participate, with procure-ment of an ultimate product as the long-term payoff. In any case, the committeebelieves that $1.5 to $2.5 billion canserve as a guide for the cost of develop-ing two to three antiviral drugs.

For comparison, the economic cost ofthe 2001 anthrax attacks, including $220million to decontaminate mail facilitiesin Maryland and New Jersey, have beenestimated in the billions (20). The re-lease of an infectious agent such assmallpox would certainly have an evengreater impact. For example, one esti-mate of the cost in 2003 of SARS forthe world economy as a whole (includ-ing both the direct costs experienced byhealth-care systems and the indirectcosts associated with disrupted com-merce, travel, and education) was closeto $40 billion (21).

The average time from initiation of adiscovery program to approval of a newdrug frequently is cited as 10–15 years(22). Streamlining and knowledge-sharingmight reduce this time somewhat, andanimal rather than human efficacy trialsmight reduce it further. The committeethus believes that 7–10 years is a reason-able estimate of the time needed toachieve an approved product. Productionand stockpiling times would add to thisinterval before treatment of an exposedpublic could be realized.Current and expected initiatives. NIAID hasreleased two broad research solicitationsfor product development that require amajor commitment from industry. One,‘‘Biodefense Partnerships: Vaccines,Adjuvants, Therapeutics, Diagnostics,and Resources,’’ involves partnershipsbetween pharmaceutical companies andthe government (23). Academic investi-gators also may be involved. Continuedfunding of such partnership projects de-pends on meeting predetermined mile-stones. Three of the 28 awards made infiscal year 2003 were for the develop-ment of poxvirus therapeutics, includingone to inhibit poxvirus phosphatases,another exploring the efficacy of high-titer vaccine immune globulin, and athird for development of an orallyadministered lipid-ether conjugate of

cidofovir. Cidofovir is an acyclic nucleo-side phosphonate with broad-spectrumactivity against a large number of vi-ruses, and it is likely to be useful intreating smallpox infections and compli-cations from smallpox vaccination. Be-cause the characteristics of cidofovir asa poxvirus therapeutic are not well un-derstood, it is important for NIH to di-rect step-two and step-three programs insearch of other treatments, engaging themost competitive biotechnology andpharmaceutical companies as rapidly aspossible.

A second research solicitation thatcurrently targets industry is the ‘‘SmallBusiness Biodefense Program’’ (24).One of the 19 fiscal year 2003 awardstargets poxvirus therapeutics. This pro-gram cannot support poxvirus researchat our most substantial pharmaceuticalor biotechnology companies, becausethey do not qualify as ‘‘small.’’

The BioShield initiative, proposed inlegislation that is before Congress at thetime of this writing, may help to addressstep four. BioShield is intended to pro-vide funds for creating and stockpilingcountermeasures against potential bio-weapons; smallpox is foremost amongthese. Its central concept is that NIHshould be able to assure pharmaceuticalcompanies that there will be a market ifan effective product is discovered, devel-oped, and delivered. As emphasizedabove, companies are unlikely to investthe human and technologic resourcesnecessary for the discovery of importantnew smallpox therapeutics without in-centives such as those that BioShieldmay offer. Even if properly funded insteps two, three, and four of a long-termantipoxvirus discovery and developmentprogram, companies must be able toexpect with confidence that there willbe a market for a successful product.BioShield, as the committee understandscurrent plans, envisions that fundingfor step four will be included in prene-gotiated procurement agreements, con-tingent on successful development ofcandidate drugs that result from directfunding of steps two and three. Thecommittee believes this to be a usefulmodel.Liability Issues. In addition to giving thepharmaceutical and biotechnology in-dustries adequate research support anda guarantee of an eventual market forthe antismallpox therapeutics that theydevelop, the issue of product liabilityalso should be addressed. As discussedin a National Academies report oncountermeasures to biowarfare agents(25), the concern over liability risksmight significantly deter some firmsfrom applying their expertise to the de-velopment of new antivirals. That report

noted that, under the Homeland Secu-rity Act of 2002 (Public Law 107–296),manufacturers of the current smallpoxvaccine would be deemed employees ofthe Public Health Service for the pur-poses of liability claims (other than forgross negligence, willful misconduct, orillegal conduct) should the vaccine beadministered in response to a declara-tion by the Secretary of the DHHS. Thismodel and others should be consideredby the Congress to address the issue ofliability in the development of newsmallpox antivirals.

Attracting Academic Investigators. Anoverriding goal is not simply to increasethe quantity of work but also to attractresearchers of high quality and strongcommitment. We distinguish mecha-nisms for making rapid, short-termprogress from those designed for longer-term enhancement of poxvirus research.A good way to start will be to partnervaccinia experts with established investi-gators who have expertise in other fieldsof biology. To expedite such interac-tions, NIH should immediately provideadministrative supplements to estab-lished investigators for specific collabo-rative projects with clearly defined,short-term objectives. The supplementsshould stipulate exchange of technologyor personnel between laboratories todisseminate expertise as rapidly as possi-ble. To provide for longer-term collabo-rations, coinvestigator-initiated grantscould pair poxvirologists with outstand-ing investigators in cell biology, struc-tural biology, chemistry, immunology,and other fields.

For the longer term, attracting new,young investigators into poxvirus-related research must have high prior-ity. The committee proposes threemechanisms by which this goal mightbe accomplished: (i) exchange of stu-dents and postdoctoral fellows betweenlaboratories of complementary exper-tise through specially awarded 1- or2-year fellowships; (ii) increased sup-port for institutional training grants inviral pathogenesis, specifically to focusattention of graduate programs andtheir students on relevant fields; and(iii) a prestigious, competitive fellow-ship program (appointing, for example,a group of NIH Fellows in Pathogene-sis) to encourage the most creativeyoung investigators to build careers invirology. To promote a career commit-ment, the fellowship award should in-clude a 3-year postdoctoral fellowshipfollowed by an additional 3 years offaculty support that facilitates an effec-tive transition into an independentposition.

Other training opportunities should


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take advantage of proven mechanisms,such as advanced courses at the ColdSpring Harbor Laboratory and theMarine Biological Laboratory at WoodsHole. Those courses reach both new andestablished investigators and are gener-ally 1–3 weeks long (some are longer)with a combination of lectures and prac-tical laboratories. Enrollment is small(15–20 investigators) and includes a mix-ture of graduate students, postdoctoralfellows, young faculty, and the occa-sional experienced investigator seekingto venture into a new field. Generally,one or two course directors are selectedto establish a curriculum, invite otherfaculty to give lectures or set up labora-tory exercises, and negotiate with thehost facility for appropriate space andphysical resources. For a course on pox-viruses, lectures might cover the virallife cycle or host–virus interactions atthe cellular and organismal level, and itwould inform the prospective research-ers about the available reagents. By us-ing vaccinia virus as a model, laboratoryexercises might introduce participants tobasic techniques of virology, includingselection and mutagenesis, as well ascell-based and in vitro assays of viralactivity. Organizing two such courseseach summer, for example, one empha-sizing the molecular and cellular biologyof poxviruses, the other emphasizingpathogenesis and virus–host interac-tions, would provide a relatively inex-pensive, rapid way for poxvirologists torecruit and identify committed collabo-rators, as well as to generate new inter-actions and ideas.

The Role of Federal Agencies and the Re-search Infrastructure. If the academic initi-atives outlined in the preceding para-graphs are to succeed, an enhancedresearch infrastructure will be vital. Aprincipal role of NIH is to foster academicresearch and industrial development, asoutlined in the preceding paragraphs.NIAID has recently established eight re-gional centers of excellence in biodefenseand awarded one-time grants for two na-tional biocontainment laboratories [bio-safety level (BSL)-3�4]. These new centersand laboratories should become majorresources for the biology communitystudying poxviruses. The unique, in-houseresearch at CDC also will play a key rolein the development of new drug candi-dates against smallpox, and CDC mustmaintain its poxvirus program at a highlevel.Repositories, databanks, and compound librar-ies. Research tools and infrastructure. Acommon theme throughout the commit-tee’s discussions was a need to go be-yond a simple repository of virus strainsto the creation of centralized resources,

pulled together by an organization thatcould commission the production of re-agents (chemical libraries, monoclonalantibodies against an extensive set ofproteins, and so on), oversee their judi-cious distribution, and streamline thefulfillment of regulatory requirements.Obtaining suitable permits is a neces-sary but major barrier to undertakingresearch on certain pathogens. In ourview, the central body should facilitatethe process of obtaining approvals fromWHO and CDC for using such reagentsas variola-virus genes and variola-specific proteins. For example, it mightset up a system for approving and peri-odically inspecting participating labora-tories so that individual investigatorsneed deal with only one agency.

Informatics tools. Some of the infor-mational infrastructure that can enablenew academic laboratories to engage inpoxvirus research already is being de-veloped. Support and expansion ofthese efforts would expedite their util-ity. The Poxvirus Bioinformatics Re-source Center provides a relationaldatabase of poxvirus genomic se-quences and their annotation and anal-ysis, web-based data-mining andsequence-analysis tools, software forgenome analysis, a poxvirus literatureresource, a repository of poxvirus spe-cies and strains at the American TypeCulture Collection (ATCC), and a dis-cussion forum. This database couldprofitably be augmented with informa-tion on mutants and phenotypes, aswell as on pharmacologic effectors.

Biodefense repository. A contract hasrecently been awarded to ATCC forcreation of a repository of existing lab-oratory tools for biodefense-relatedresearch based on the AIDS repositorymodel. Current plans envision the dis-tribution of poxvirus strains, proteins,and DNA, but the extent of what willbe distributed has not yet been estab-lished. A priority should be placed onthe development and distribution ofnew poxvirus tools, including polyclonaland monoclonal antibodies to viral ORFs,glutathione S-transferase (GST)-fusionexpression clones (pGEX), Gateway PCRclones (validated by sequence), GFP-labeled ORFs, and tandem affinity purifi-cation (TAP)-tagged proteins. Vacciniavirus is the model system of choice, onwhich most of this work should focus. Butfor biochemical studies and ultimately forscreening, consideration should be givento generating a set of variola-virus PCRproducts for individual ORFs, (such asin Gateway vectors); here, of course, itwill be important to observe regulatorysafeguards.

Compound libraries. Specific inhibitorsof poxvirus targets will be useful as

probes of the viral life cycle. Academicscientists with access to the relevant ex-pertise and to biochemical or cellularassays generally lack access to suitablechemical libraries. The committee pro-poses that NIH consider establishing aNational Compound Library, whichwould expand on existing compoundrepositories, such as those held by theNational Cancer Institute. A NationalCompound Library should contain 0.5–1million drug-like compounds, and suffi-cient amounts should be collected toensure longevity and a capacity to servequalified groups throughout the country.Such groups would be expected to havehigh-throughput screening capability,secondary assay technologies for deter-mining the selectivity of identifiedcompounds, and expertise in syntheticorganic chemistry. Because the cost ofmaintaining such a library could be sub-stantial, it should probably serve otherprograms in addition to antiviral-drugdiscovery.

Coordination of facilities. The NIAIDregional centers of excellence are anexcellent way to coordinate and imple-ment the committee’s recommendationsfor national facilities. A group that in-cludes the principal investigators ofthese centers and of the regional andnational laboratories is being establishedby NIH to oversee their activities. Thisbody, and the regional centers them-selves, can provide mechanisms for car-rying out the recommendations in thepreceding paragraphs. The availability ofBSL-3 and BSL-4 facilities in the re-gional and national laboratories alsomight afford the opportunity for estab-lishing training programs in BSL-3�4techniques and in the administrativeprocedures, protocols, and permissionsneeded to work with variola genes.CDC facilities. CDC in Atlanta and Vektorin Novosibirsk are the only laboratorieswhere research on variola is permittedby WHO. CDC has two BSL-4 facilities,each of which can accommodate up to16 monkeys (and larger numbers of ro-dents). No other BSL-4 agent work canbe conducted in a laboratory where vari-ola virus is in use, and the current andplanned studies on variola virus occupyfully one-half of CDC’s space capacityfor such pathogens. The BSL-4 facilitiesare available no more than 8 months peryear, because of the need for preventivemaintenance and safety procedures. Atpresent, some needed equipment (such astelemetry, flow cytometers, and massspectrometers) is unavailable in the maxi-mal containment laboratory, and there isinsufficient room to accommodate it.Some of these deficiencies may be cor-rected when new maximal containmentlaboratories, now under construction,


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come online at CDC in 2–3 years. How-ever, because of the pivotal role thatthis laboratory will have in testing prom-ising drug candidates against variola vi-rus, CDC must establish long-term goalsfor its poxvirus research program andtake measures to ensure its continuingfunction.

Regulatory Questions. FDA rules on efficacyevaluation. The Animal Efficacy Rule,finalized by FDA in 2002, allows FDAto approve drugs and vaccines when it isnot ethical or feasible to conduct humanefficacy studies, as is the case for asmallpox antiviral drug. FDA require-ments are not clear, however, becausethe animal models of smallpox have notbeen studied in the detail that usuallyaccompanies work on other viral dis-eases. At the very least, scientists mustunderstand the pathophysiology of thedisease process and define the mecha-nism by which a proposed countermea-sure would reverse it. The degree ofdetail required is pivotal to proper studydesign. The few monkey model studiescompleted to date measured many pa-rameters, some of which may not benecessary. For example, data are col-lected on viral quantization, lesioncounts, hematology, clinical chemistry,cytokine profiles, cDNA microarrayanalysis, and cellular and humoral im-munologic responses. In general, FDAprefers end points based on mortalityrather than morbidity. A morbidity endpoint more closely approximates humansmallpox (up to 40% mortality), butthese experiments require the use ofmany animals. Uniform mortality (theanimal survives or it does not) can beachieved only with a model that resem-bles hemorrhagic, rather than common,smallpox. These issues are complex, butFDA must resolve them soon so that theexperimental design for determinationof efficacy can be defined.WHO restrictions. WHO guidelines limitthe extent of experimental work that ispermitted on variola virus. Those guide-lines, formulated over 2 decades ago,were based on the assumption thatsmallpox had been eradicated and thatresearch on the virus, its genes, and itsproteins should be restricted to guardagainst the possibility of an accidentalrelease. Do recent events necessitate areappraisal? If other countries or otherentities covertly hold stocks of the virus,should WHO reassess its rules? Theseissues deserve debate in an internationalsetting because a smallpox outbreakwould create an international healthcrisis.

Continued compliance with interna-tional accords is, of course, essential,although the restriction of all variola

research to the two WHO collaboratingcenters does limit research in the field(as was intended). Unfortunately, thesite in Russia (Vektor) is under-equipped and inadequate to the task ofcontemporary studies, and CDC facili-ties are presently unequipped to conductdetailed pathogenesis studies in animalsin BSL-4 containment. Variola viruscould be handled safely in any BSL-4facility that has a proven track record ofbiosafety in the use of pathogens forwhich no vaccines or therapies are avail-able. Thus, one question is whether theimportance of the research program rec-ommended in the first part of this re-port and the evaluation of the likelihoodof covert stocks outside Vektor andCDC make it necessary to discuss alter-ing the two-site restriction. The commit-tee suggests that CDC explore withWHO whether other qualified laborato-ries under its control might be deemedpart of its site. It also suggests that CDCdedicate a larger fraction of its BSL-4facilities to variola virus research andmove other programs to sites elsewhere.

WHO currently prohibits any geneticmanipulation of the variola genome.The prohibition extends to the incorpo-ration of reporter genes, such as GFP,which would greatly facilitate high-throughput antiviral drug screens. GFPhas been introduced into numerous vi-ruses without affecting virulence. Awhite paper proposing GFP insertionhas been presented to WHO on two oc-casions, most recently in November2002. It has not yet been considered bythe WHO orthopoxvirus committee,however, in part because of concern thatapproval of this proposal would openthe door to more worrisome or danger-ous genetic manipulations.

WHO restrictions pertain not only tovariola virus but also to possession andexpression of its genes. No laboratoryoutside CDC and Vektor may holdmore than 20% of the total genome orhold even that if it is using other or-thopoxviruses. Although currently notpermitted, the introduction of singlevariola virus genes into vaccinia viruscould be useful for testing antivirals andmonoclonal antibodies in small-animalmodels without the hazard posed byworking with variola virus.

In summary, WHO should be encour-aged to reevaluate its rules in light of anindependent estimation of the bioterroristthreat and of the contemporary scientificbreakthroughs that could be exploited toaddress it.

Implementation. Implementing the vari-ous components (academic and indus-trial, national and international) of asmallpox antiviral program will require

a consistent sense of urgency and afocused decision-making process. Ahigh-level advisory and oversight panel,analogous to the AIDS Vaccine Re-search Working Group, should be cre-ated immediately, reporting to theheads of NIH, CDC, and any otherfederal agencies involved in this effort(including branches of the Departmentof Homeland Security). Membershipon this committee should representexperience in drug discovery and de-velopment, poxvirus expertise at thehighest level, and international effortsin smallpox antiviral research. Its first,urgent task will be to establish a specifictimeline for drug discovery and develop-ment, including prioritization of currentlyfeasible targets and approaches. A secondimportant task will be to work out a blue-print for an effective international effort.

Other Issues. Some regulatory, political,and ethical issues are important butoutside the scope of discussions of thisgroup. The potential for misuse of theknowledge gained in further poxvirusresearch is of concern, just as thepotential for dual use of knowledge isof concern in much other scientificresearch. Who should decide the aus-pices under which particular experi-ments should be done? How should theresults be analyzed and disseminated? TheNational Academies report ‘‘Biotechnol-ogy Research in an Age of Terrorism:Confronting the ‘Dual Use’ Dilemma’’presents a scholarly discussion of some ofthe issues (26).

Another question has to do with howregulatory restrictions are enforced.Suitably talented researchers alwayshave options in their choice of prob-lems to study. A climate of apprehen-sion about how even inadvertent lapsesmight be handled will drive away pre-cisely the sorts of investigators whomwe hope to attract into research onpoxviruses or other topics relevant tobiodefense. Apparent lapses in correctresearch practice should be dealt withby the oversight mechanisms that al-ready govern handling of pathogensand hazardous or radioactive materials,not by the criminal justice system.

For additional reading, see refs. 27and 28.

Summary of Recommendations. Based onthe preceding discussion, the commit-tee presents three categories in whichactions taken by NIH and otherbranches of the federal governmentwould enhance the prospects for thedevelopment of antiviral drugs againstsmallpox. These are: (i) establishingnovel contractual relationships withdrug companies (Recommendation 1,


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below); (ii) invigorating poxvirus re-search in academic and governmentlaboratories (Recommendations 2, 3,and 4, below); and (iii) forging consen-sus on the criteria for safety and effec-tiveness of bioweapons countermea-sures and on the regulations that willgovern their development (Recommen-dations 5, 6, and 7, below).

Recommendations1. Pharmaceutical and biotechnology companyengagement.

a. We propose an immediate focus oncurrently identifiable therapeutic tar-gets that are essential enzymes ofpoxviruses (see Table 3). To gain theparticipation of major companies, wesuggest the following program:

i. direct contact between seniorgovernment official and leaders ofmajor pharmaceutical and bio-technology companies to initiateand encourage participation;

ii. targeted solicitation of proposalsto carry out the screening ofchemical libraries beginning withcurrently identifiable targets andelaboration of an early lead dis-covery phase, followed by federalsupport of preclinical develop-ment in those companies thatpresent the most promising dis-covery-phase data; and

iii. support of full clinical develop-ment of the best preclinicalcandidates through negotiatedguarantees of purchase contingenton meeting predetermined prod-uct specifications.

b. Successful engagement of industrythrough the process just outlined willrequire:

i. high-level planning and oversightof the process by the advisorypanel proposed in Recommenda-tion 6;

ii. new procedures for rapid, insight-ful review, including participationof individuals with extensive expe-rience in drug discovery and de-velopment;

iii. addressing the issue of indemnifi-cation, through legislation to limitthe liability of companies thatdevelop smallpox antivirals underthis program; and

iv. consortium arrangements thatpermit companies to share infor-mation and thereby reduce risks.

c. Therapeutics against smallpox willneed to be licensed under the FDA’sAnimal Efficacy Rule. To promotesound application of this rule, NIH,the U.S. Army Medical Research In-stitute of Infectious Diseases, CDC,and the scientific community at large

should engage in research on appro-priate animal models for poxvirusinfections and on the kinds of studiesneeded to show efficacy of antiviraldrugs. The FDA itself also shouldreceive sufficient funding to supportits own work on these issues, so thatthe criteria for licensing smallpox an-tivirals can be established as early aspossible.

2. Focused, short-term basic research. Rapidprogress toward deeper understandingof poxvirus biology will result from ac-tive collaborations between academiclaboratories already engaged in poxvirusresearch and those with specific biologicor technologic expertise. NIH shouldsupport established investigators, nation-ally and internationally, to work withpoxvirologists on well defined, short-term projects, in which technology andpersonnel are exchanged, and also onlonger-term, coinvestigator-initiated col-laborations. The structural biology ofpoxvirus proteins, the cell biology ofpoxvirus infection, and the interactionbetween poxviruses and the host im-mune system are three areas in whichsuch collaborations should be encour-aged. Establishing the credibility of ani-mal models for variola infection is also ahigh priority.3. Recruiting and training a new cohort ofyoung investigators. Long-term progresswill require ‘‘new blood,’’ and researchinitiatives should be designed with thispoint in mind.

a. NIH should attract new talent intopoxvirology by granting 1- to 2-yearfellowships that permit students andpostdoctoral scientists to move betweenlaboratories with complementary ex-pertise and by awarding additional in-stitutional training grants in viralpathogenesis.

b. NIH should support one or more lab-oratory courses in poxvirus researchat sites such as Cold Spring Harbor,NY, or Woods Hole, MA.

c. A high-profile fellowship (NIHFellow in Viral Pathogenesis) shouldbe established to support the mostoutstanding students during postdoc-toral studies and their first few yearsof faculty research.

4. Centralized resources. NIH, with the col-laboration of CDC, should establish re-positories and databanks. Large librariesof chemical compounds will be particu-larly useful for academic investigatorswho have the experience and facilities toundertake large-scale screening. NIHand CDC also should create an accessi-ble central mechanism for facilitatingWHO and CDC approvals and for coor-dinating access to the use of national

laboratory facilities. The research cen-ters of excellence being established byNIAID’s Biodefense Program can pro-vide mechanisms for responding to someof these needs.5. WHO restrictions. NIH and CDC shouldencourage WHO to reevaluate its prohi-bition of certain manipulations of vari-ola virus that would greatly facilitatenovel screening methods, for example,the introduction of reporter genes intovariola virus and the use of variola virusgenes in other vectors. Mechanismsshould be developed for undertakingsuch studies in CDC poxvirus researchlaboratories. CDC also should explorewith WHO whether other qualified lab-oratories under its control might bedeemed part of its site and otherwisetake measures to ensure the continuing,vigorous function of its poxvirus re-search effort.6. Implementation. In view of the impor-tance of including expert scientific guid-ance throughout the steps recommendedin this report, a high-level advisory andoversight panel, analogous to the AIDSVaccine Research Working Group,should be created immediately, report-ing to the heads of NIH, CDC, and anyother federal agencies involved in thiseffort. Membership on this committeeshould represent experience in drugdiscovery and development, poxvirusexpertise at the highest level, and inter-national efforts in smallpox antiviral re-search. Its first, urgent task will be toestablish a specific timeline for drug dis-covery and development, including pri-oritization of currently feasible targetsand approaches. A second importanttask will be to work out a blueprint foran effective international effort (seeRecommendation 7).7. International participation. The prospectof an intentional release of the variolavirus is a global concern. Research onbioweapon countermeasures thereforeshould involve broad international col-laborations. There should be no unduerestrictions on the participation of non-U.S. citizens in work at U.S. laborato-ries, and grant and contract fundingshould extend, where appropriate forsupport of the best science, to laborato-ries outside the U.S.

We extend our gratitude to the individualswho attended the National Academies’ June15–16, 2003, workshop on ‘‘Transforming Bi-ological Information into New Therapies:Smallpox Antivirals,’’ and acknowledgeNIAID for its support of this activity. In ad-dition, we thank A. Mahmoud, D. Bloom,and E. Cordes for their useful insights on thepharmaceutical industry, and T. Caskey, M.Collett, J. Henney, N. Nathanson, R. Moyer,V. Sato, P. Palese, and P. Traktman for help-ful comments on the draft of this article.


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discovery of antivirals against smallpox · virus (smallpox virus) and molluscum contagiosum virus,...

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