Discovery of antivirals against smallpox

Stephen 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, 2101 Constitution 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, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10021; jDepartment of Pathology, Harvard Medical School, NRB, 77 Avenue Louis Pasteur, Boston, MA 02115; kDepartment of Cell Biology, The Scripps Research Institute, 10550 North 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, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Stephen C. Harrison, May 21, 2004

mallpox, a devastating infectious Whatever the likelihood of covertly dopoxviruses has a restricted and spe- disease dreaded throughout much held variola virus stocks, an intentional cific host array (Table 2). Humans are of recorded history, is caused by release of the virus would pose a serious the sole hosts of two poxviruses, variola the variola virus, a member of health threat and would probably pro- virus (smallpox virus) and molluscum Sthe poxviridae family. In the 20th cen- voke a global health crisis. The lethality contagiosum virus, although many mem- tury alone, smallpox deaths worldwide of the disease (up to 40%) and its ease bers of Orthopoxvirus, Parapoxvirus, and numbered in the millions. In 1980, after of transmissibility place variola virus at Yatapoxvirus can infect both animals and an intensive program of immunization the top of CDC’s list of high-threat humans (Table 2, zoonotic viruses). with vaccinia virus, a related but rela- (Category A) agents. For that reason, Vaccinia virus is the virus used in the tively nonpathogenic virus, the World the federal government rapidly in- variola virus vaccine, and it is widely Health Organization (WHO) declared creased its support of research related used as a model poxvirus in the labora- the disease eradicated. By 1983, all to the discovery of antiviral drugs tory. Its origins are obscure. known stocks of variola virus were in against smallpox. In addition to provid- The genomes of 30 poxviruses have two WHO collaborating centers: the ing potential therapy for infected peo- been completely sequenced, including U.S. Centers for Disease Control and ple, the availability of antiviral drugs several strains of variola and vaccinia Prevention (CDC) in Atlanta and (after could decrease the risks associated with viruses. At least two variants of variola a transfer in 1994) the Russian State the smallpox vaccine by providing a virus are known, and they cause two Research Center of Virology and Bio- treatment for vaccine-associated compli- forms of smallpox: variola major, with technology (the Vektor Institute) in cations. Ultimately, the development of a case fatality rate of 30–40%, and Novosibirsk. The WHO Committee on antiviral drugs against smallpox could variola minor, with a much reduced deter rogue states and terrorists from Orthopoxvirus Infections voted on sev- fatality rate of Ϸ1%. At the genome releasing variola virus because its impact eral occasions to recommend destruc- level, the two variants are very similar. would be diminished. Moreover, the tion of the stocks, but each time the They differ in Ϸ2% of the roughly same new therapies are likely to be use- decision was deferred to permit more 185,000 unique DNA nucleotides; es- ful in other poxvirus diseases, such as research on live variola virus. A 1999 sentially all of the encoded proteins monkeypox. National Academies report summarized are nearly identical. These comparisons suggest that the high fatality rate asso- and assessed scientific needs for live Scientific Opportunities variola virus (1). ciated with variola major may be at- Introduction to Poxviruses.n The poxvirus tributable to a small number of genetic The concern that undeclared stocks of family. Poxviruses are the largest known determinants, and they point to our variola virus might exist and that they animal viruses, with Ϸ200 distinct genes ignorance of the causes of poxvirus might be used as a bioterrorist weapon (5). They are DNA viruses that replicate pathogenesis in general and of variola (2) was heightened in late 2001 by the entirely in the cytoplasm. Thus, a subset virus pathogenesis in particular. Eradi- deliberate release of Bacillus anthracis, of their gene products carries out the the agent of anthrax, in the weeks after functions that are essential for the vi- the September 11, 2001, attacks. That ruses to be independent of the host-cell Abbreviations: WHO, World Health Organization; CDC, U.S. concern prompted a voluntary national- nucleus. The other viral gene products Centers for Disease Control and Prevention; IMV, intracel- preparedness effort to vaccinate health- lular mature vaccinia virion; EEV, extracellular enveloped use or modulate an astonishingly wide virion; ITR, inverted terminal repetition; IEV, intracellular care workers, first responders, and array of host-cell and immune-system enveloped virion; CEV, cell-associated virion; UDG, uracil members of the military against small- processes. DNA glycosylase; IV, immature virion; NIH, National Insti- pox. However, given the substantial side Poxviruses infect most vertebrates and tutes of Health; SARS, severe acute respiratory syndrome; effects, the risks associated with the FDA, Food and Drug Administration; DHHS, Department of invertebrates, causing a variety of dis- Health and Human Services; NIAID, National Institute of smallpox vaccine, and the absence of eases of veterinary and medical impor- Allergy and Infectious Diseases; BSL, biosafety level. information about an imminent bioter- tance. The one large family (Poxviridae) bTo whom correspondence should be addressed. E-mail: rorist attack, vaccination was not ac- has two main subfamilies, the chor- [email protected]. cepted by all members of those groups, dopoxvirinae, which infect vertebrates, nAdapted with permission from ref. 4 (Copyright 2003, ASM nor was it recommended for the general and the entomopoxvirinae, which infect Press). public by the government (3). insects (Table 1). Each of the chor- © 2004 by The National Academy of Sciences of the USA

11178–11192 ͉ PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 RMTEAAEY PERSPECTIVE ACADEMY: THE FROM

Table 1. The poxvirus family: Poxviridae Subfamily Genus Type species

Chordopoxvirinae Orthopoxvirus Vaccinia virus (vertebrate hosts) Parapoxvirus Orf virus Avipoxvirus Fowlpox virus Capripoxvirus Sheeppox virus Leporipoxvirus Myxoma virus Suipoxvirus Swinepox virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus 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) cation of smallpox occurred at a time rus biology is derived largely from stud- biology are in place to dissect the virus– of limited knowledge of the molecular ies of this virus. The tools available for cell interactions. and cellular nature of the relevant host its study now include large collections of Poxvirus replication cycle. Poxviruses have a defense. Thus, during the times when it temperature-sensitive mutant strains as complex structure. Fig. 1A shows an was possible to study human infections, well as recombinant strains with specific electron micrograph of a cross section the molecular tools for dissecting the genes under inducible regulation. Sev- of the infectious intracellular mature host response were lacking. eral of the known gene products, such vaccinia virion (IMV) and a schematic Vaccinia virus has become the model as its enzymes, can be expressed and summary of the virion. The nature of the of choice, and our knowledge of poxvi- studied in vitro, and new tools of cell membrane envelope surrounding the IMV

Table 2. Hosts of and infections caused by chordopoxvirinae Animal Virus reservoir Geographic distribution Transmission to other hosts

Genus Molluscipoxvirus Molluscum contagiosum Humans Worldwide No transmission to other hosts Genus Orthopoxvirus Zoonotic viruses Monkeypox virus* Squirrels West and Central Africa Monkeys, humans Vaccinia virus Unknown Worldwide Humans, buffaloes, rabbits, cows Buffalopox virus† Unknown Asia Buffaloes, humans Camelpox virus Camels Africa, Asia Camels Cowpox virus‡ Rodents Europe, Asia Cats, cows, zoo animals, humans Elephantpox virus Unknown Asia Elephants, humans Nonzoonotic viruses Variola virus Humans Previously worldwide Only humans Volepox virus Voles Western United States None Ectromelia virus Rodents Europe None Raccoonpox virus Raccoons Eastern United States None Skunkpox virus Skunks Eastern United States None Uasin Gishu disease virus Unknown East Africa Horses Taterapox virus Gerbils West Africa None Genus Parapoxvirus Zoonotic viruses Bovine papular stomatitis virus Cattle Worldwide Humans Orf virus Sheep Worldwide Ruminants, humans Pseudocowpox virus (milker’s nodules) Cattle Worldwide Humans Sealpox virus Seals Worldwide Humans Nonzoonotic viruses Auzduk disease virus Camels Africa, Asia None Parapoxvirus of red deer in New Zealand Deer New Zealand None Chamois contagious ecthyma virus Chamois — — Genus Yatapoxvirus Tanapox virus Rodents East and Central Africa Monkeys, humans Yaba 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 Fig. 1. Vaccinia virus, a representative poxvirus: virion structure (A) and genome organization with an expanded view of the HindIII D restriction enzyme fragment (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 detailed description. [Reproduced with permission from ref. 4 (Copyright 2003, ASM Press).] particle remains controversial. Some argue contain no obvious helical or icosahedral AT-rich and lie at the ends of 12-kbp that there are two closely opposed lipid nucleocapsid. A second infectious particle, inverted terminal repetition (ITR) ele- bilayer membranes; others argue that the extracellular enveloped virion (EEV), ments that contain short direct repeats there is one. (The answer has important contains an additional lipid bilayer mem- and several ORFs. The genome se- implications for viral entry and matura- brane that is wrapped around the entire quence reveals some 185 putative pro- tion, as will be discussed below). This IMV particle. tein-coding sequences and provides a IMV envelope forms the outer boundary The genomes of poxviruses are dou- detailed genetic map. of a 300-Å-thick surface layer that sur- ble-stranded DNA molecules. Fig. 1B The basis for naming the viral pro- rounds the inner core, which often ap- summarizes information obtained from teins is illustrated by the coding se- pears dumbbell-shaped. The outer row of the completely sequenced Copenhagen quences of HindIII fragment D, which reproducibly observed knobs on the core and WR strains of vaccinia virus. The are shown in the expanded view in Fig. wall has been termed the palisade. The 191-kbp genome is a double-stranded 1B. The ORFs are depicted by the ar- core contains the double-stranded viral DNA molecule whose ends are co- rows indicating their orientation in the DNA genome and virion enzymes, includ- valently connected by single-stranded genome, and each is named with the ing DNA-dependent RNA polymerase hairpin loops of 101 nucleotides. The letter of the HindIII fragment in which and RNA-processing enzymes. Pox virions sequences that form the hairpins are the first ATG of the reading frame lies,

11180 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. followed by a number indicating its or- der and its orientation, either left (L) or right (R). The identities of the encoded proteins, where known, also are listed. As illustrated in Fig. 1B, the vaccinia coding sequences are densely packed, and the template for RNA synthesis may be present in either strand. The se- quences encoding structural proteins and essential enzymes are clustered in roughly the central 120 kb, whereas genes encoding virulence proteins, host- range proteins, or immunomodulators are found predominantly near the ends. This arrangement is also true for all other poxviruses. Proteins that fulfill different functions are synthesized during different phases of the infectious cycle. For example, the mRNA capping enzyme, uracil DNA glycosylase (UDG), and most RNA polymerase subunits are products of early genes, whereas the genes encoding structural proteins are expressed only during the late phase of infection. In addition, a small but distinct class of proteins including the late transcription factors is encoded by intermediate Fig. 2. The single-cell reproductive cycle of vaccinia virus. The entry and replication of an EEV are genes. illustrated. RNA molecules are green. See Poxvirus replication cycle for a detailed description of each Even though they are DNA viruses, illustrated step. [Reproduced with permission from ref. 4 (Copyright 2003, ASM Press).] poxviruses replicate in the cytoplasm. Accordingly, they are only minimally dependent on the host cell for DNA and tors, which can induce proliferation of synthesized by the cellular translation RNA replication, and they encode their neighboring host cells, or are proteins machinery (Fig. 2, step 11), the assem- own proteins for these processes. A sim- that counteract host immune defense bly of progeny virus particles begins. plified version of the single-cell repro- mechanisms. The synthesis of early pro- The viral membrane proteins are ungly- ductive cycle of vaccinia virus is illus- teins also induces a second uncoating cosylated, and the role of cellular mem- trated in Fig. 2. EEV entry is illustrated reaction in which the core wall opens branes in early stages of assembly is in Fig. 2, step 1. The mechanisms by and a nucleoprotein complex containing controversial (Fig. 2, step 12). which either the IMV or the EEV infec- the genome is released from the core The initial assembly reactions result in tious forms of vaccinia virus attach to (Fig. 2, step 5). This core disassembly formation of the immature virion (Fig. and enter susceptible host cells are not leads to cessation of viral early gene 2, step 13), which is a spherical particle well understood and will be constrained expression. Other early proteins catalyze delimited by a membrane that may be by the number of membranes enveloping the replication of the viral DNA ge- acquired from an early compartment of the virus. After fusion of the viral and nome (Fig. 2, step 6). Newly synthesized the cellular secretory pathway. This vi- cellular membranes, primary uncoating viral DNA molecules can serve as tem- rus particle matures into the brick- takes place, and the viral core is re- plates for additional cycles of genome shaped IMV (Fig. 2, step 14), which is leased into the cytoplasm. All later steps replication (Fig. 2, step 7), and they are released only on cell lysis (Fig. 2, step in the infectious cycle take place in this the templates for transcription of viral 15). However, the particle can acquire a cellular compartment. The core con- intermediate-phase genes (Fig. 2, step second, double membrane from a trans- tains, in addition to the viral genome, 8). The activation of transcription of Golgi or early endosomal compartment the viral DNA-dependent RNA poly- intermediate genes also requires specific to form the intracellular enveloped merase, the ‘‘initiation’’ proteins necessary viral proteins (the products of early virion (IEV) (Fig. 2, step 16). The IEVs for specific recognition of the promoters genes) that confer specificity for inter- move to the cell surface on microtubules of viral early genes, and several RNA- mediate promoters on the viral RNA where fusion with the plasma membrane processing enzymes that modify viral polymerase, as well as a host-cell pro- forms cell-associated virions (CEV) transcripts. On release into the host-cell tein (Vitf2) that relocates from the (Fig. 2, step 17). These CEV induce an cytoplasm, the core synthesizes viral infected cell nucleus to the cytoplasm. actin polymerization that promotes a early mRNAs (Fig. 2, step 2), which ex- The proteins encoded by intermediate direct transfer to surrounding cells (Fig. hibit the features typical of cellular mRNAs (Fig. 2, step 9) include those 2, step 18); they can also dissociate from mRNAs and are translated by the cellu- necessary for transcription of late-phase the membrane as EEV. lar protein-synthesizing machinery (Fig. genes (Fig. 2, step 10). The latter genes Although studies of viral entry and 2, step 3). Approximately half of the encode the proteins from which virions morphogenesis have revealed striking viral genes are expressed during this are built as well as the virion enzymes mechanisms by which the viral proteins early phase of infection. and other essential proteins, such as the interact with the actin and microtubule Some early proteins are secreted from early initiation proteins, that must be cytoskeleton, they also have left us with the cell (Fig. 2, step 4) and have se- incorporated into virus particles during several unresolved puzzles in membrane quence similarity to cellular growth fac- assembly. Once these proteins are biogenesis. In addition, there is an ex-

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11181 Dual-specificity phosphatases (DSPs) participate in a number of cellular sig- naling pathways. Their activities are rel- evant to various disease states, and there has been substantial work on their pharmacology. The DSP domain of H1 is small (171 aa), however, and lacks unique structural features. There are also highly conserved ‘‘vaccinia H1-like’’ homologues in humans. Thus, a better candidate for an antiviral might be the F10 dual-specificity protein kinase, which also is critical for the earliest steps of virus morphogenesis. It has no sequence similarity to known cellular kinases, with the exception of a 110-aa region of a protein known as human H1 protein phosphatase. The F10 protein sequences in variola and monkeypox viruses are 98% identical. The I7 cysteine protease, encapsi- dated in the virus particle, processes the major virion core proteins at a cleavage motif, AlaGly1X. A loss in functional- Fig. 3. Schematic representation of selected poxvirus immunomodulators and regulators of apoptosis. ity of the enzyme leads to arrest of Proteins shown in red represent poxvirus proteins; host proteins are shown in black and gray. Secreted virion morphogenesis at a point after poxvirus viroceptor proteins (top row) function as soluble or cell surface decoys that bind host-cell cytokines or chemokines in the cell exterior. Poxvirus virokines also are secreted; they function as agonistic the formation of spherical immature or antagonistic ligands for host cellular receptors (middle row). A number of poxvirus proteins function particles. This protein of 432 aa is 99% inside the cell to modulate apoptosis, cytokine processing, and host range (red proteins in cell interior). identical in variola and monkeypox vi- [Reproduced with permission from ref. 7 (Copyright 2003, Annual Reviews, www.annualreviews.org).] ruses; it appears to have only a very distant relationship to the cellular SUMO-specific protease Ulp1 and to tensive molecular dialogue between the promptly. These criteria narrow the field adenovirus and African swine fever viral gene products and the vertebrate of candidate targets considerably. So far, virus (ASFV) proteases. host’s innate and adaptive immune sys- the atomic structures of five poxvirus pro- The A22 Holliday junction (HJ) re- tems, as illustrated by the known targets teins have been determined: topoisomer- solvase is a small, 187-aa, metal-depen- ase I, cap-specific 2ЈO-methyltransferase of many of the virus-encoded immune ͞ dent nuclease. The encapsidated enzyme modulators and regulators of apoptosis poly(A) polymerase stimulatory subunit, is synthesized late in infection and par- (Fig. 3). These include secreted ‘‘viro- complement-control protein, CrmA im- ticipates in the resolution of concate- kines’’ that mimic host cytokines, se- mune modulator, and an inhibitor of the meric DNA replication intermediates creted ‘‘viroceptors’’ that mimic host RNA-dependent protein kinase. A pox- before genome packaging. A distant rel- cytokine receptors, and intracellular virus structural-biology initiative would ative of the bacterial HJ resolvase RuvC proteins that interfere with apoptosis be a valuable component of an orga- and fungal mitochondrial resolvase and signaling pathways. nized effort to discover inhibitors of vi- In short, it is clear that poxviruses ral enzymes. The enzymes listed in Cce1, the nuclease is peculiar to poxvi- constitute an underexploited tool for Table 3 have at least some of the de- ruses and extocarpus siliculosus virus; probing fundamental processes in both sired characteristics. no human orthologues have been identi- cell biology and immunology. By taking Drugs that target topoisomerases and fied. Little is known about possible HJ advantage of this tool, we will certainly cause them to damage DNA are among nuclease inhibitors and their potential as increase our understanding of important the most successful therapeutics devel- drugs. biological mechanisms; we are also very oped for both infectious disease (quino- Uracil DNA glycosylase (UDG) is likely to discover unexpected ways to lones) and cancer (etoposide, adriamycin, a 218-aa protein that is required for control poxvirus infections. and camptothecins). Indeed, drug screen- DNA replication. The UDG has an ing ‘‘in silico’’ (that is, by computer essential role in viral DNA replication Routes to New Antiviral Therapeutic modeling) is already under way for pox- that is independent of its catalytic ac- Agents: Poxvirus Enzymes as Candidate virus topoisomerase I, a 314-aa mono- tivity. However, viruses encoding a cat- Drug Targets. Essential viral enzymes mer comprising two domains joined by a alytically inactive UDG are attenuated have frequently proved to be good tar- flexible molecular hinge. A recent study in vivo. gets for antiviral drugs (for example, indicates that the enzyme is not abso- The poxvirus capping enzyme is a herpesvirus thymidine kinase, HIV re- lutely essential but has an important multisubunit complex responsible for verse transcriptase and protease, and role in enhancing early transcription in adding a 7-methyl guanosine ‘‘cap’’ to influenza neuraminidase). Initial efforts the virus core (13). the 5Ј end of viral messenger RNA. Al- to identify inhibitors should therefore H1 protein phosphatase, a dual-speci- though they accomplish the same func- focus on enzymes that have essential ficity cysteine phosphatase encapsidated tion, there are important biochemical roles in poxvirus replication and for in the virus particle, is required in order differences between the capping com- which atomic structures have been for the encapsidated viral genome to be plexes of poxviruses and those of their determined or can be determined transcribed by the viral transcriptase. mammalian hosts.

11182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. 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 factor A22 Holliday junction resolvase D4 Uracil DNA glycosylase D5 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 (ϩJ3) Poly(A) polymerase [catalytic (ϩstimulatory)] D1 (ϩD12) mRNA capping enzyme [catalytic (ϩstimulatory)] H6 Topoisomerase I H1 Protein phosphatase (dual specificity) Protein modification and virus assembly (Fig. 2, step 14) F10 Protein kinase (dual specificity) I7 Cysteine protease E10, A2.5, G4 Thiol oxidases

The virion encapsidated RNA poly- DNA polymerase. The DNA polymerase host-cell lines, but the identity of host- merase (RNAP) holds great potential as inhibitor cidofovir inhibits poxvirus cell receptors and the routes of entry a drug target. This nine-subunit enzyme, DNA polymerases, including that of var- into the cytosol remain unknown (Fig. packaged in the viral core, transcribes iola virus, and it has been shown to 4, step 1). There are two infectious genes expressed early in replication. have antiviral effects in animal models. forms of the virus, IMV and EEV, with Many of the subunits are not found in The drug currently is used to treat mol- either one or two (IMV) or two or three cellular RNA polymerases. RNAP ap- luscipoxvirus infection in AIDS patients (EEV) lipoprotein bilayers surrounding pears to have a unique initiation mecha- and is recommended by CDC for use in the nucleoprotein core. The identity of nism that requires early transcription treating complications of vaccination putative viral fusion proteins is un- factors (ETFs) found only in poxviruses. with existing smallpox vaccines. known, but the unusual arrangement of Attempts to target pox RNAP so far Three conserved poxvirus thiol oxi- the poxvirus envelope may imply that its have taken two approaches. The first is dases, only distantly related to cellular fusogens are mechanistically distinct to use small molecules to block pro- proteins, are required for formation of from more familiar viral fusion proteins. moter recognition or ATP hydrolysis by the disulfide bonds in several viral mem- Identification and characterization of ETFs. The second is to devise an brane proteins. Deletion or mutation of the entry, fusion, and uncoating mecha- ‘‘mRNA poison’’: here the virus is pro- any of these proteins prevents virus as- nisms of poxviruses should therefore vided with a substrate analogue (for sembly. Thus, each of these redox pro- broaden our understanding of the gen- example, adenosine N1 oxide) that is teins would provide a good target for eral mechanism of membrane fusion, incorporated into the viral mRNA by developing an antiviral drug. which is central to a wide variety of crit- poxvirus RNAP, but the mRNA cannot ical processes in cells. be translated into viral proteins. Cell Biology of Poxviruses. Six aspects of Regulation and organization of DNA replica- The poxvirus poly(A) polymerase is a poxvirus research are particularly ripe tion, transcription, and translation. Poxviruses heterodimer composed of catalytic and for investigation and offer substantial are unique among animal DNA viruses in processivity subunits. The processivity opportunities for discovery. They are: (i) carrying out DNA replication and tran- subunit, for which the atomic structure the mechanism of entry into host cells; scription in the interphase cytoplasm. Ex- is known, also methylates the ribose of (ii) the regulation and intracellular orga- pression of the viral genome thus includes mRNA caps and works as a transcrip- nization of DNA replication, DNA tran- opportunities for types of regulation unfa- tion elongation factor. scription (RNA synthesis), and translation miliar elsewhere in animal cell biology The E9 DNA polymerase, required (protein synthesis); (iii) the viral manipu- and hence opportunities to identify spe- for DNA replication, acts in concert lation of host-cell membrane traffic; (iv) cific antiviral targets. Only the bare essen- with accessory proteins to attain effi- the viral subversion and exploitation of tials of the regulation of poxvirus gene cient processive synthesis. Accessory host-cell signaling pathways; (v) the viral expression are understood. Promoter se- proteins include the A20 protein, D4 exploitation of motor proteins for move- quences, the virus-encoded RNA poly- UDG, and possibly others. The D5 ment within a cell and for propulsion merase, and transcription factors have NTPase and the viral SSB (the I3 protein) toward another cell; and (vi) the mecha- been identified (14), but the mecha- also are essential for viral replication. The nism of cell-level host-range restriction. nisms of transcription initiation, elonga- NTPase has homology to helicases, al- This list of topics indicates the wide ar- tion, and termination are not known in though it is not yet known to act in this ray of advanced scientific expertise that any molecular detail. Data from in vitro capacity. Viral DNA polymerases are should be used in future studies of pox- studies indicate a role of host proteins established drug targets, as exemplified virus biology to increase our under- in transcription, but no supporting in by azidothymidine (AZT), which inhibits standing of the infectious process. Fig. 4 vivo data have been reported. It has the HIV reverse transcriptase, and acy- highlights features of poxvirus cell biol- long been known that intermediate and clovir, which is efficiently phosphory- ogy and some of the unanswered ques- late transcripts have nontemplated 5Ј lated by the herpes simplex virus viral tions for future research. poly(A) leader sequences, but their role thymidine kinase, resulting in a triphos- Entry into host cells. Vaccinia virus can has not been clarified. After virus infec- phate that preferentially inhibits viral invade and replicate in a wide array of tion, the turnover of all mRNAs is

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11183 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 together with 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 of membrane crescents, an early stage in viral assembly. The derivation of the membrane from either the endoplasmic reticulum (ER) or intermediate compartment (IR) is uncertain, although regularly spaced small spikes can be seen on the outer membrane (arrows). [Reproduced with permission from ref. 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).] (Upper Right)Afluorescent micrograph of actin tail formation (green) juxtaposed to and triggered by cell-surface associated CEV (red), which function to propel the virus particle away from the cell and͞or into adjacent cells. 4Ј,6-Diamidino-2-phenylindole (DAPI) staining (blue) reveals a large viral factory adjacent to 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).] greatly increased by an unknown mecha- test bed for the screening of potential an- components have in scaffolding or di- nism. With regard to DNA replication, tiviral drugs. [For a review of other such recting them? several essential proteins have been systems, see Bell et al. (15)]. Viral manipulation of host-cell membrane traf- identified by genetic screens, but their There are cytologic hints that poxvi- fic. To what extent does the virus exploit functions, except for that of the DNA ruses establish some type of membrane- host-cell structures for organization of its polymerase, are not well defined (5). enclosed ‘‘virus factory’’ in the assembly process, and to what extent does Other features of poxvirus DNA replica- cytoplasm where replication takes place it remodel the cell scaffold to fit its needs tion deserve attention. The linear DNA (Fig. 4, step 2). What is the nature of (16)? Host membrane alterations are genome is terminated by hairpin loops this putative compartment, and how closely coupled to spatial targeting, trans- at each end and is thus a covalently does the virus set it up? Transcription port, and assembly of virus-particle com- closed, single-stranded DNA. How does and translation of at least some viral ponents. An early event in morphogenesis replication begin on this template? A products are spatially controlled in the is the appearance within virus-factory re- focused effort on developing an in vitro host cell, and some kinds of directed gions of membrane ‘‘crescents,’’ which DNA replication system composed of transport must take place during virus- may be closely apposed lipid bilayers de- purified poxvirus proteins capable of particle assembly (Fig. 4, step 4). What rived from endoplasmic reticulum mem- replicating double-stranded DNA tem- is the molecular basis of these processes, brane (Fig. 4, step 3). How do the viral plates could provide a particularly fruitful and what roles do host-cell structural proteins target, recruit, and grossly rear-

11184 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. range the host membranes to form these licating virus and the host cell should sis, with a focus on primate models (7). unusual structures, and how are viral ge- reveal novel targets for antiviral therapy. Fortunately, the available human arrays nomes delivered to and inserted into appear to be useable for nonhuman- them? Is there any relationship of crescent Research Needs in Pathogenesis and Host primate samples as well. formation to autophagy? At later stages, Response. Animal models. Because of the If an animal model (such as monkey- the viral particles undergo a second layer host-range specificity and containment pox in cynomolgus monkeys) is deemed of membrane wrapping, this time recruit- requirements of variola virus, closely an adequate surrogate for variola virus, ing trans-Golgi or endosomal membranes related orthopoxviruses that replicate in it will be of the utmost importance to (Fig. 4, step 5). Analysis of mutants and rodents (such as cowpox virus, ectrome- track the various immune characteristics recombinant strains has enabled identifi- lia virus, and vaccinia virus, including known to correlate with B cell and T cation of viral proteins that are involved the rabbitpox strain) are usually used for cell activation and with emergence of in many of these membrane manipula- studies of viral pathogenesis. Monkey- memory B and T cells. That goal will tions, creating a diverse molecular toolkit, pox in the cynomolgus monkey is the require establishment (in the BL4 facil- both for exploring cell membranes and best nonhuman-primate model, although ity for variola virus and in appropriate organelles and for providing a diverse ar- large doses of virus are required. Mon- containment facilities for other poxvi- ray of targets for inhibitors. keypox virus is a select agent (a hazard- ruses) of the appropriate cell-sorting Viral exploitation of host-cell signaling path- ous biological agent subject to safety instruments, allowing researchers to ways. Before the assembled particle be- regulations), and Biosafety Laboratory 3 readily obtain the relevant leukocyte comes infectious, an essential virally en- facilities are required, but primate mod- subsets from infected animals (B cells, coded protease must cleave several els are essential for testing vaccines and T cells, macrophages, dendritic cells, proteins in a process that is coupled to therapeutics at late stages in their devel- and so forth). Similarly, obtaining puri- condensation of the core. A virus-specific opment. More accessible and less expen- fied leukocyte subsets in this way will be group of thiol transferase enzymes also sive animal models will be critical for essential for evaluating cytokine produc- catalyzes formation of disulfide bonds on rapid work at earlier stages, and devel- tion. Wherever a mouse model can be the intracytoplasmic tails of virus mem- opment of realistic animal models for used, advantage can be taken of the brane, enabling this oxidation to occur in orthopoxvirus infection and disease outstanding sets of reagents that are an apparently reducing environment (17). (such as models for which the innocu- available (antibodies, nucleic acids, and The initial, immature particle, the imma- lum required for infection is not heroic knockout animals). Those experiments ture virion (IV), is noninfectious, but the and for which the endpoint is not neces- should be started now. sarily death) should be emphasized. Re- Immunotherapeutics. Protective human or final result of the maturation steps, the search across the entire spectrum of humanized monoclonal antibodies to IMV, is both highly infectious and stable. poxvirus genera should be strongly en- variola virus for use in immunotherapy How does morphogenesis occur, and what couraged, because the history of virol- should be developed with modern tech- molecular and mechanical events are in- ogy has shown that key insights into one nologies. These antibodies would be di- volved? How is protein processing by group of viruses often come from the rected to the viral surface proteins and cleavage and disulfide formation linked to study of an apparently divergent group. possibly to secreted products. Many of the morphologic alterations? What key Proteomics. There is a clear need for a the antibodies would crossreact with differences determine the infectivity of complete analysis of the structural and other orthopoxviruses and could be the IMV as opposed to the IV? nonstructural proteins of variola virus tested in animal models. The perceived Viral exploitation of motor proteins. The as- and related orthopoxviruses supported importance of a ‘‘cytokine storm’’ (in sembled particles use microtubule mo- by the requisite bioinformatics tools. which the production and release of tors to get to the perinuclear region for Neither infectious variola virus nor in- inflammatory cytokines and stress medi- envelopment in the Golgi membrane tact variola virus DNA can be trans- ators overwhelms the host) as a major (Fig. 4, step 4) and subsequently move ferred from the two authorized smallpox determinant of the pathologic condition along microtubules to the cell surface facilities, but WHO regulations allow offers further opportunities for immune (Fig. 4, step 6) (18). Moreover, budded other laboratories to work with DNA intervention (for example, with tumor extracellular virions still attached to the representing up to 20% of the variola necrosis factor antagonists). cell membrane direct assembly of intra- virus genome in nonpoxvirus vectors. A Safer vaccines. Further knowledge of or- cellular actin bundles at positions just coordinated international effort to study thopoxvirus proteins, the mechanisms of beneath their membrane attachment variola virus proteins therefore will be infection, and the correlates of immu- point, driving formation of a membrane necessary if we are to acquire a proper nity may enable the design of safer stalk that bears the virion (CEV) at its understanding of the viral proteome. smallpox vaccines. Possible approaches tip (Fig. 4, step 7). How do viral pro- Viral and cellular determinants of host re- include the development of attenuated teins establish these assemblies? Which sponse. As is the case with many other strains of vaccinia virus, recombinant proteins are essential for spread of the viruses, little is known with certainty proteins, and vectors. Any antipoxvirus infection in a tissue? about immune characteristics that corre- therapeutics developed will make it pos- Mechanism of host-range restriction in vitro late with protective immunity against sible to treat the rare complications of and in vivo. There are a number of mo- variola viruses or other poxviruses. Con- vaccination, making vaccinations even lecularly defined mutant vaccinia virus sequently, all aspects of both the innate safer. strains that replicate in some mamma- and acquired immune response must be lian lines but not in others. Those studied. It is important to define the Discussion. Naturally occurring variola strains provide an opportunity to define innate immune response to a poxvirus virus has been eradicated from the the role of host-cell factors in viral rep- infection, including cytokine production planet. Indeed, the last reported case of lication and morphogenesis. These and regulation of cellular receptors in- natural smallpox occurred in Somalia in mutant viruses also may facilitate the volved in activation of elements of the 1977, long before recent advances in development of safer vaccines. innate immune system. The capabilities molecular biology, cell biology, and im- Understanding the complex molecular of modern DNA-array technology munology would have allowed research- interactions that occur between the rep- should be brought to bear on this analy- ers to study the variola virus and the

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11185 pathogenesis of human infection in fine goal in mind. In the second part of this are formulated here in the context of detail. Studies on the closely related report, Incentives, Logistics, and Policies, U.S. institutions (for example, NIH) vaccinia virus have provided a relatively we consider the organizational require- the committee urges that its proposals thorough outline of poxvirus replication ments for achieving each of those objec- be taken in an international context and in mammalian cells, but a great deal tives and propose specific ways to meet that the initiatives recruit broad inter- remains to be understood and little is them. national participation. known about how the human immune There is a pressing need for new anti- system responds to variola virus. Given viral drugs and new vaccines, whether to Engaging the Pharmaceutical and Biotech- the virulence of this virus (up to 40% counter potential bioterrorist threats, to nology Industries. The importance of indus- mortality) and its ability to spread in a treat HIV, or to deal with emerging trial participation at early stages in the discov- population, the consequences of an in- pathogens like severe acute respiratory ery of smallpox antivirals. If the discovery tentional release of variola virus could syndrome (SARS) coronavirus. Collabo- and development of antiviral therapeu- be devastating. Official stocks of the rations that bring poxvirologists together tics to treat poxvirus infections is to be virus are closely held, but it is not with outstanding scientists in other a high priority, it is essential that phar- known whether undeclared stocks exist, fields of biology are not only likely to maceutical and biotechnology companies so it is difficult to assess the current de- accelerate the search for ways to control be engaged from the outset. Academic gree of risk. Safer vaccines and therapeu- infection by variola virus but also to researchers eventually may acquire the tics that can mitigate the consequences of produce discoveries relevant to diverse resources, equipment, compound librar- infection would together provide a strong pathogens, including other DNA viruses ies, and experience required to partici- deterrent to any intentional release. Effec- and intracellular bacteria. Such collabora- tive development of such deterrents will tions will also expand our understanding pate successfully in drug discovery, but benefit greatly from a deeper understand- of the fundamental cellular processes that that process will certainly take years. ing of the biology of poxviruses and of poxviruses exploit. Meanwhile, there are many pharma- how they interact with their hosts. There In the broadest sense, directing the ceutical and biotechnology companies is an enormous range of opportunities: attention of a larger number of out- already poised to discover poxvirus- because variola is a large virus, it requires standing scientists with expertise in cell specific antiviral drugs, beginning with many specific viral-encoded proteins to biology, structural biology, computa- the candidate targets listed in Table 3. infect humans, each of which represents a tional biology, and chemical biology to These companies currently have the potential target for an antiviral drug. the complexities of viral pathogenesis equipment and compound libraries re- We recommend a research-and-devel- can be a fruitful effort for increasing quired to screen more than a million opment response at three levels. First, both national and international security. compounds per month against any drug we propose an immediate focus on cur- In the long run, a well conceived, con- target peculiar to poxviruses. Moreover, rently identifiable therapeutic targets, certed program to discover treatments these for-profit companies have the ex- particularly essential poxvirus-encoded for smallpox (should it reemerge) will pertise needed to assess the attributes enzymes, through a combination of aca- provide models for how to motivate in- or deficits of compounds emerging at demic and industrial research. The phar- terdisciplinary groups to solve other ma- early stages from high-throughput drug maceutical industry has enormous jor problems in public health. It will screens; to synthesize the hundreds of experience in the discovery, design and likewise demonstrate how to create ef- analogues of early drug candidates re- development of enzyme inhibitors, and fective incentives for participation of the quired to move such compounds from each of the enzymes listed in Table 3 is biotechnology and pharmaceutical in- the early ‘‘hit’’ stage into bona fide drug a potential antiviral target. We recom- dustries in the production of new types leads; to perform the pharmacologic mend that the National Institutes of of protective agents against viruses and and toxicologic assessments essential for Health (NIH) use the mechanisms out- other threat agents, whether these are the generation of substantive, preclinical lined in Incentives, Logistics, and Policies introduced intentionally or through nat- drug leads; to perform the detailed, below (see especially Recommendation ural processes. regulated preclinical studies needed for 1) to initiate, both in pharmaceutical Incentives, Logistics, and Policies submission of an investigational new and biotechnology companies and in drug (IND) application to the Food and some collaborating academic laborato- It is not known whether there are stocks Drug Administration (FDA); and to ries, immediate work on each of these of variola virus other than those sanc- perform clinical trials at the safety and enzymes. Second, we suggest that rapid, tioned by the WHO or whether small- efficacy stages of drug development. short-term progress toward deeper un- pox will someday become a weapon The success of both traditional phar- derstanding of poxvirus biology will re- used by terrorists or rogue states. If the maceutical companies and biotechnology sult from active collaborations between latter were to occur, the reemergence of government or academic laboratories smallpox would create a major interna- companies in discovering, developing, already engaged in poxvirus research tional health crisis. To counter the po- and marketing novel therapeutics spe- and others with specific biologic or tech- tential use of variola virus as a weapon cific for the treatment of HIV demon- nologic expertise. As examples of such and to protect the public in the event of strates the power of these organizations collaborations, we recommend NIH- an intentional release, a concerted inter- for producing clinically valuable antiviral sponsored initiatives in the structural national effort is needed to tackle the therapeutics. Poxviruses offer a far biology of poxvirus proteins, in the cell scientific challenges described in the broader spectrum of therapeutic targets biology of poxvirus infection, and in the first part of this report and to translate than does HIV, and it is reasonable to interaction between poxviruses and the the results of that research into new expect that finding poxvirus-specific host immune system. Third, we observe therapeutic candidates. drugs will be less demanding than the that longer-term progress will require It is important to recognize that the challenges presented by HIV. We there- recruitment and training of a new co- eradication of naturally occurring small- fore believe that, with the engagement hort of outstanding young investigators, pox was achieved through a global ef- of appropriate pharmaceutical and bio- and we suggest that the initiatives just fort. Therefore, although many of the technology groups, clinically valuable listed be designed with this long-term recommendations related to those issues products will emerge.

11186 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. A program for engaging the pharmaceutical this process is that it tends to reward are implemented will need to respond to industry. Engaging pharmaceutical and the first company to bring forward a the uncertainties and surprises of discov- biotechnology companies in a poxvirus drug rather than the one that produces ery and development. The concept of drug-discovery effort will require signifi- the best drug. large, long-term contractual relation- cant changes in the ways that NIH, An alternative strategy for step four ships with for-profit companies is rela- CDC, and other federal agencies have would be to create a ‘‘public pharma- tively new to NIH and to the Depart- traditionally interacted with industry. ceutical company,’’ either specifically for ment of Health and Human Services First, because the government is likely development of smallpox antivirals or (DHHS). However, the National Insti- to be the sole purchaser of any prod- more broadly for other bioweapons tute of Allergy and Infectious Diseases ucts, their development will require in- countermeasures. Such a course would, (NIAID) has had some experience in centives and resources at critical stages. however, require the duplication of this type of decision-making, both Second, the way in which programs and talent and resources already available through its AIDS vaccine research effort proposals are evaluated and reviewed within the pharmaceutical industry. The and through the creation of the intramu- will require advisory structures and pro- long timescale for drug development ral Vaccine Research Center. cedures quite different from the peer- also might be inappropriate for a single The high-level advisory and oversight review system that works well for aca- mission-oriented facility. panel described below in the section Im- demic research awards. It would be desirable to have the par- plementation should have a major role in The committee suggests the following ticipation of pharmaceutical companies guiding the interaction of NIH and in- outlines of a program to meet the chal- of various sizes in the discovery and de- dustry. In addition, the leadership of lenges of industry incentives. First, se- velopment process. The committee NIH and DHHS should examine how nior government health officials should found, in discussions with individuals other branches of the federal govern- make a direct and open invitation to the acquainted with both large pharmaceuti- ment, such as the Department of De- highest levels of pharmaceutical and cal companies and smaller biotechnol- fense, interact with the private sector, as biotechnology management. A widely ogy firms, that engaging companies in it develops its own model for such col- announced, high-profile program would these two categories might require dif- laboration. allow companies to be seen as contribut- ferent approaches. For example, the NIH will be defining goals and steer- ing to an important public-health re- contract program outlined as steps two ing research. It therefore will be essen- sponse. Second, NIH should provide and three in the preceding paragraph tial to develop effective modalities of broad support for the early stages of would probably work better for smaller internal and external review so that NIH drug discovery, beginning with currently companies, whereas larger firms might can respond rapidly to proposals under identifiable targets and continuing as respond better to a single contract cov- the multistep program outlined above basic research uncovers new targets and ering both these steps, specifying appro- and so that it can have effective input novel strategies. Third, several awards priate milestones for continuation. from individuals with experience and (perhaps five or six in total) should be The government also might consider good judgment in practical drug discov- made to companies that have demon- assembling a consortium of pharmaceu- ery and development. The mechanisms strated their ability to deliver the most tical and biotechnology companies to of peer review used by NIH to set prior- attractive preclinical candidate drugs, share knowledge as discovery proceeds. ities for support of academic science will derived from work in the previous stage. In this setting, the exchange of informa- be inadequate for evaluating programs These awards would support accelerated tion would reduce individual opportu- that require large, long-term funding of preclinical drug development, including nity costs. Moreover, if one company private-sector research and develop- ‘‘good manufacturing process’’ produc- were to discover a particularly favorable ment, both because conventional tion, full evaluation of drug metabolism synthetic scaffold or series of com- academic review groups tend to be rela- and pharmacokinetics, toxicology and pounds that inhibit a specific target, it tively conservative and because the animal efficacy tests, and appropriate would make sense for the entire com- contract mechanism requires different regulatory compliance leading to an in- munity to focus on it. Alternatively, it qualities than a grant. vestigational new drug submission to the might be appropriate to distribute work The committee notes that Anthony FDA. on different targets among different Fauci, director of NIAID, already has The fourth step of the proposed anti- participating companies. Because the broached the subject of antivirals poxvirus drug development program government is not the customary market against smallpox with the heads of sev- includes clinical studies and preparation for pharmaceutical companies, and be- eral leading pharmaceutical and biotech- of a new drug application (NDA) for cause knowledge-sharing might lead to nology firms, initiating what we define approval by the FDA. This will be the advances in understanding of antivirals here as step one. most expensive phase of the process, in general, pharmaceutical companies As a rough measure of investment and it is particularly for this step that might be more willing than usual to ex- costs, the committee offers the following new government͞industry relationships change information. The issues of anti- outline. Recent research, based on a will need to emerge. One potential trust regulation and intellectual property survey of a large number of companies, strategy, which is incorporated in the protection raised by such information- finds that development of a single new pending BioShield legislation (see be- sharing would need to be considered, drug costs Ϸ$800 million (year 2000 low), involves the concept of a guaran- but if the program were deemed a suffi- dollars) in out-of-pocket expenses, capi- teed market, with prenegotiated prices ciently critical national priority, they talized to the point of marketing ap- contingent on successful product devel- should not be insurmountable. proval (19). This estimate, which may be opment (as defined by FDA licensing) The committee believes that in setting the high end of a spectrum of possible within a fixed time frame. Key decisions up and implementing a program such as values, includes the costs of failed candi- will need to be made about the negotia- the one outlined above, it is especially dates at various stages in the develop- tion of a guaranteed market and price important that wise decisions be made ment process. Clinical trials will be less and about the timing and amount of by using the best scientific judgment and expensive in the case of smallpox antivi- government support during the develop- astute management considerations. rals, because efficacy tests will involve ment process. A possible drawback of Moreover, the ways in which programs animal rather than human subjects, but

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11187 the cost savings might be offset by the cidofovir. Cidofovir is an acyclic nucleo- noted that, under the Homeland Secu- requirements of a shorter-than-average side phosphonate with broad-spectrum rity Act of 2002 (Public Law 107–296), timeline. The committee therefore chose activity against a large number of vi- manufacturers of the current smallpox to use the estimate $800 million for the ruses, and it is likely to be useful in vaccine would be deemed employees of overall cost of developing a single drug. treating smallpox infections and compli- the Public Health Service for the pur- Procurement, stockpiling, and renewal cations from smallpox vaccination. Be- poses of liability claims (other than for costs are, of course, additional. (Re- cause the characteristics of cidofovir as gross negligence, willful misconduct, or newal costs refer to expenses required a poxvirus therapeutic are not well un- illegal conduct) should the vaccine be to ascertain potency of stockpiled mate- derstood, it is important for NIH to di- administered in response to a declara- rials and to replace them as needed.) It rect step-two and step-three programs in tion by the Secretary of the DHHS. This remains to be determined whether con- search of other treatments, engaging the model and others should be considered tract support for all or a substantial most competitive biotechnology and by the Congress to address the issue of fraction of the $800 million would offset pharmaceutical companies as rapidly as liability in the development of new opportunity costs sufficiently to induce possible. smallpox antivirals. companies to participate, with procure- A second research solicitation that ment of an ultimate product as the long- currently targets industry is the ‘‘Small Attracting Academic Investigators. An term payoff. In any case, the committee Business Biodefense Program’’ (24). overriding goal is not simply to increase believes that $1.5 to $2.5 billion can One of the 19 fiscal year 2003 awards the quantity of work but also to attract serve as a guide for the cost of develop- targets poxvirus therapeutics. This pro- researchers of high quality and strong ing two to three antiviral drugs. gram cannot support poxvirus research commitment. We distinguish mecha- For comparison, the economic cost of at our most substantial pharmaceutical nisms for making rapid, short-term the 2001 anthrax attacks, including $220 or biotechnology companies, because progress from those designed for longer- million to decontaminate mail facilities they do not qualify as ‘‘small.’’ term enhancement of poxvirus research. in Maryland and New Jersey, have been The BioShield initiative, proposed in A good way to start will be to partner estimated in the billions (20). The re- legislation that is before Congress at the vaccinia experts with established investi- lease of an infectious agent such as time of this writing, may help to address gators who have expertise in other fields smallpox would certainly have an even step four. BioShield is intended to pro- of biology. To expedite such interac- greater impact. For example, one esti- vide funds for creating and stockpiling tions, NIH should immediately provide mate of the cost in 2003 of SARS for countermeasures against potential bio- administrative supplements to estab- the world economy as a whole (includ- weapons; smallpox is foremost among lished investigators for specific collabo- ing both the direct costs experienced by these. Its central concept is that NIH rative projects with clearly defined, health-care systems and the indirect should be able to assure pharmaceutical short-term objectives. The supplements costs associated with disrupted com- companies that there will be a market if should stipulate exchange of technology merce, travel, and education) was close an effective product is discovered, devel- or personnel between laboratories to to $40 billion (21). oped, and delivered. As emphasized disseminate expertise as rapidly as possi- The average time from initiation of a above, companies are unlikely to invest ble. To provide for longer-term collabo- discovery program to approval of a new the human and technologic resources rations, coinvestigator-initiated grants drug frequently is cited as 10–15 years necessary for the discovery of important could pair poxvirologists with outstand- (22). Streamlining and knowledge-sharing new smallpox therapeutics without in- ing investigators in cell biology, struc- might reduce this time somewhat, and centives such as those that BioShield tural biology, chemistry, immunology, animal rather than human efficacy trials may offer. Even if properly funded in and other fields. might reduce it further. The committee steps two, three, and four of a long-term For the longer term, attracting new, thus believes that 7–10 years is a reason- antipoxvirus discovery and development young investigators into poxvirus- able estimate of the time needed to program, companies must be able to related research must have high prior- achieve an approved product. Production expect with confidence that there will ity. The committee proposes three and stockpiling times would add to this be a market for a successful product. mechanisms by which this goal might interval before treatment of an exposed BioShield, as the committee understands be accomplished: (i) exchange of stu- public could be realized. current plans, envisions that funding dents and postdoctoral fellows between Current and expected initiatives. NIAID has for step four will be included in prene- laboratories of complementary exper- released two broad research solicitations gotiated procurement agreements, con- tise through specially awarded 1- or for product development that require a tingent on successful development of 2-year fellowships; (ii) increased sup- major commitment from industry. One, candidate drugs that result from direct port for institutional training grants in ‘‘Biodefense Partnerships: Vaccines, funding of steps two and three. The viral pathogenesis, specifically to focus Adjuvants, Therapeutics, Diagnostics, committee believes this to be a useful attention of graduate programs and and Resources,’’ involves partnerships model. their students on relevant fields; and between pharmaceutical companies and Liability Issues. In addition to giving the (iii) a prestigious, competitive fellow- the government (23). Academic investi- pharmaceutical and biotechnology in- ship program (appointing, for example, gators also may be involved. Continued dustries adequate research support and a group of NIH Fellows in Pathogene- funding of such partnership projects de- a guarantee of an eventual market for sis) to encourage the most creative pends on meeting predetermined mile- the antismallpox therapeutics that they young investigators to build careers in stones. Three of the 28 awards made in develop, the issue of product liability virology. To promote a career commit- fiscal year 2003 were for the develop- also should be addressed. As discussed ment, the fellowship award should in- ment of poxvirus therapeutics, including in a National Academies report on clude a 3-year postdoctoral fellowship one to inhibit poxvirus phosphatases, countermeasures to biowarfare agents followed by an additional 3 years of another exploring the efficacy of high- (25), the concern over liability risks faculty support that facilitates an effec- titer vaccine immune globulin, and a might significantly deter some firms tive transition into an independent third for development of an orally from applying their expertise to the de- position. administered lipid-ether conjugate of velopment of new antivirals. That report Other training opportunities should

11188 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. take advantage of proven mechanisms, pulled together by an organization that probes of the viral life cycle. Academic such as advanced courses at the Cold could commission the production of re- scientists with access to the relevant ex- Spring Harbor Laboratory and the agents (chemical libraries, monoclonal pertise and to biochemical or cellular Marine Biological Laboratory at Woods antibodies against an extensive set of assays generally lack access to suitable Hole. Those courses reach both new and proteins, and so on), oversee their judi- chemical libraries. The committee pro- established investigators and are gener- cious distribution, and streamline the poses that NIH consider establishing a ally 1–3 weeks long (some are longer) fulfillment of regulatory requirements. National Compound Library, which with a combination of lectures and prac- Obtaining suitable permits is a neces- would expand on existing compound tical laboratories. Enrollment is small sary but major barrier to undertaking repositories, such as those held by the (15–20 investigators) and includes a mix- research on certain pathogens. In our National Cancer Institute. A National ture of graduate students, postdoctoral view, the central body should facilitate Compound Library should contain 0.5–1 fellows, young faculty, and the occa- the process of obtaining approvals from million drug-like compounds, and suffi- sional experienced investigator seeking WHO and CDC for using such reagents cient amounts should be collected to to venture into a new field. Generally, as variola-virus genes and variola- ensure longevity and a capacity to serve one or two course directors are selected specific proteins. For example, it might qualified groups throughout the country. to establish a curriculum, invite other set up a system for approving and peri- Such groups would be expected to have faculty to give lectures or set up labora- odically inspecting participating labora- high-throughput screening capability, tory exercises, and negotiate with the tories so that individual investigators secondary assay technologies for deter- host facility for appropriate space and need deal with only one agency. mining the selectivity of identified physical resources. For a course on pox- Informatics tools. Some of the infor- compounds, and expertise in synthetic viruses, lectures might cover the viral mational infrastructure that can enable organic chemistry. Because the cost of life cycle or host–virus interactions at new academic laboratories to engage in maintaining such a library could be sub- the cellular and organismal level, and it poxvirus research already is being de- stantial, it should probably serve other would inform the prospective research- veloped. Support and expansion of programs in addition to antiviral-drug ers about the available reagents. By us- these efforts would expedite their util- discovery. ing vaccinia virus as a model, laboratory ity. The Poxvirus Bioinformatics Re- Coordination of facilities. The NIAID exercises might introduce participants to source Center provides a relational regional centers of excellence are an basic techniques of virology, including database of poxvirus genomic se- excellent way to coordinate and imple- selection and mutagenesis, as well as quences and their annotation and anal- ment the committee’s recommendations cell-based and in vitro assays of viral ysis, web-based data-mining and for national facilities. A group that in- activity. Organizing two such courses sequence-analysis tools, software for cludes the principal investigators of each summer, for example, one empha- genome analysis, a poxvirus literature these centers and of the regional and sizing the molecular and cellular biology resource, a repository of poxvirus spe- national laboratories is being established of poxviruses, the other emphasizing cies and strains at the American Type by NIH to oversee their activities. This pathogenesis and virus–host interac- Culture Collection (ATCC), and a dis- body, and the regional centers them- tions, would provide a relatively inex- cussion forum. This database could selves, can provide mechanisms for car- pensive, rapid way for poxvirologists to profitably be augmented with informa- rying out the recommendations in the recruit and identify committed collabo- tion on mutants and phenotypes, as preceding paragraphs. The availability of rators, as well as to generate new inter- well as on pharmacologic effectors. BSL-3 and BSL-4 facilities in the re- actions and ideas. Biodefense repository. A contract has gional and national laboratories also recently been awarded to ATCC for might afford the opportunity for estab- The Role of Federal Agencies and the Re- creation of a repository of existing lab- lishing training programs in BSL-3͞4 search Infrastructure. If the academic initi- oratory tools for biodefense-related techniques and in the administrative atives outlined in the preceding para- research based on the AIDS repository procedures, protocols, and permissions graphs are to succeed, an enhanced model. Current plans envision the dis- needed to work with variola genes. research infrastructure will be vital. A tribution of poxvirus strains, proteins, CDC facilities. CDC in Atlanta and Vektor principal role of NIH is to foster academic and DNA, but the extent of what will in Novosibirsk are the only laboratories research and industrial development, as be distributed has not yet been estab- where research on variola is permitted outlined in the preceding paragraphs. lished. A priority should be placed on by WHO. CDC has two BSL-4 facilities, NIAID has recently established eight re- the development and distribution of each of which can accommodate up to gional centers of excellence in biodefense new poxvirus tools, including polyclonal 16 monkeys (and larger numbers of ro- and awarded one-time grants for two na- and monoclonal antibodies to viral ORFs, dents). No other BSL-4 agent work can tional biocontainment laboratories [bio- glutathione S-transferase (GST)-fusion be conducted in a laboratory where vari- safety level (BSL)-3͞4]. These new centers expression clones (pGEX), Gateway PCR ola virus is in use, and the current and and laboratories should become major clones (validated by sequence), GFP- planned studies on variola virus occupy resources for the biology community labeled ORFs, and tandem affinity purifi- fully one-half of CDC’s space capacity studying poxviruses. The unique, in-house cation (TAP)-tagged proteins. Vaccinia for such pathogens. The BSL-4 facilities research at CDC also will play a key role virus is the model system of choice, on are available no more than 8 months per in the development of new drug candi- which most of this work should focus. But year, because of the need for preventive dates against smallpox, and CDC must for biochemical studies and ultimately for maintenance and safety procedures. At maintain its poxvirus program at a high screening, consideration should be given present, some needed equipment (such as level. to generating a set of variola-virus PCR telemetry, flow cytometers, and mass Repositories, databanks, and compound librar- products for individual ORFs, (such as spectrometers) is unavailable in the maxi- ies. Research tools and infrastructure. A in Gateway vectors); here, of course, it mal containment laboratory, and there is common theme throughout the commit- will be important to observe regulatory insufficient room to accommodate it. tee’s discussions was a need to go be- safeguards. Some of these deficiencies may be cor- yond a simple repository of virus strains Compound libraries. Specific inhibitors rected when new maximal containment to the creation of centralized resources, of poxvirus targets will be useful as laboratories, now under construction,

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11189 come online at CDC in 2–3 years. How- research to the two WHO collaborating a consistent sense of urgency and a ever, because of the pivotal role that centers does limit research in the field focused decision-making process. A this laboratory will have in testing prom- (as was intended). Unfortunately, the high-level advisory and oversight panel, ising drug candidates against variola vi- site in Russia (Vektor) is under- analogous to the AIDS Vaccine Re- rus, CDC must establish long-term goals equipped and inadequate to the task of search Working Group, should be cre- for its poxvirus research program and contemporary studies, and CDC facili- ated immediately, reporting to the take measures to ensure its continuing ties are presently unequipped to conduct heads of NIH, CDC, and any other function. detailed pathogenesis studies in animals federal agencies involved in this effort in BSL-4 containment. Variola virus (including branches of the Department Regulatory Questions. FDA rules on efficacy could be handled safely in any BSL-4 of Homeland Security). Membership evaluation. The Animal Efficacy Rule, facility that has a proven track record of on this committee should represent finalized by FDA in 2002, allows FDA biosafety in the use of pathogens for experience in drug discovery and de- to approve drugs and vaccines when it is which no vaccines or therapies are avail- velopment, poxvirus expertise at the not ethical or feasible to conduct human able. Thus, one question is whether the highest level, and international efforts efficacy studies, as is the case for a importance of the research program rec- in smallpox antiviral research. Its first, smallpox antiviral drug. FDA require- ommended in the first part of this re- urgent task will be to establish a specific ments are not clear, however, because port and the evaluation of the likelihood timeline for drug discovery and develop- the animal models of smallpox have not of covert stocks outside Vektor and ment, including prioritization of currently been studied in the detail that usually CDC make it necessary to discuss alter- feasible targets and approaches. A second accompanies work on other viral dis- ing the two-site restriction. The commit- important task will be to work out a blue- eases. At the very least, scientists must tee suggests that CDC explore with print for an effective international effort. understand the pathophysiology of the WHO whether other qualified laborato- disease process and define the mecha- ries under its control might be deemed Other Issues. Some regulatory, political, nism by which a proposed countermea- part of its site. It also suggests that CDC and ethical issues are important but sure would reverse it. The degree of dedicate a larger fraction of its BSL-4 outside the scope of discussions of this detail required is pivotal to proper study facilities to variola virus research and group. The potential for misuse of the design. The few monkey model studies move other programs to sites elsewhere. knowledge gained in further poxvirus completed to date measured many pa- WHO currently prohibits any genetic research is of concern, just as the rameters, some of which may not be manipulation of the variola genome. potential for dual use of knowledge is necessary. For example, data are col- The prohibition extends to the incorpo- of concern in much other scientific lected on viral quantization, lesion ration of reporter genes, such as GFP, research. Who should decide the aus- counts, hematology, clinical chemistry, which would greatly facilitate high- pices under which particular experi- cytokine profiles, cDNA microarray throughput antiviral drug screens. GFP ments should be done? How should the analysis, and cellular and humoral im- has been introduced into numerous vi- results be analyzed and disseminated? The munologic responses. In general, FDA ruses without affecting virulence. A National Academies report ‘‘Biotechnol- prefers end points based on mortality white paper proposing GFP insertion ogy Research in an Age of Terrorism: rather than morbidity. A morbidity end has been presented to WHO on two oc- Confronting the ‘Dual Use’ Dilemma’’ point more closely approximates human casions, most recently in November presents a scholarly discussion of some of smallpox (up to 40% mortality), but 2002. It has not yet been considered by the issues (26). these experiments require the use of the WHO orthopoxvirus committee, Another question has to do with how many animals. Uniform mortality (the however, in part because of concern that regulatory restrictions are enforced. animal survives or it does not) can be approval of this proposal would open Suitably talented researchers always achieved only with a model that resem- the door to more worrisome or danger- have options in their choice of prob- bles hemorrhagic, rather than common, ous genetic manipulations. lems to study. A climate of apprehen- smallpox. These issues are complex, but WHO restrictions pertain not only to sion about how even inadvertent lapses FDA must resolve them soon so that the variola virus but also to possession and might be handled will drive away pre- experimental design for determination expression of its genes. No laboratory cisely the sorts of investigators whom of efficacy can be defined. outside CDC and Vektor may hold we hope to attract into research on WHO restrictions. WHO guidelines limit more than 20% of the total genome or poxviruses or other topics relevant to the extent of experimental work that is hold even that if it is using other or- biodefense. Apparent lapses in correct permitted on variola virus. Those guide- thopoxviruses. Although currently not research practice should be dealt with lines, formulated over 2 decades ago, permitted, the introduction of single by the oversight mechanisms that al- were based on the assumption that variola virus genes into vaccinia virus ready govern handling of pathogens smallpox had been eradicated and that could be useful for testing antivirals and and hazardous or radioactive materials, research on the virus, its genes, and its monoclonal antibodies in small-animal not by the criminal justice system. proteins should be restricted to guard models without the hazard posed by For additional reading, see refs. 27 against the possibility of an accidental working with variola virus. and 28. release. Do recent events necessitate a In summary, WHO should be encour- reappraisal? If other countries or other aged to reevaluate its rules in light of an Summary of Recommendations. Based on entities covertly hold stocks of the virus, independent estimation of the bioterrorist the preceding discussion, the commit- should WHO reassess its rules? These threat and of the contemporary scientific tee presents three categories in which issues deserve debate in an international breakthroughs that could be exploited to actions taken by NIH and other setting because a smallpox outbreak address it. branches of the federal government would create an international health would enhance the prospects for the crisis. Implementation. Implementing the vari- development of antiviral drugs against Continued compliance with interna- ous components (academic and indus- smallpox. These are: (i) establishing tional accords is, of course, essential, trial, national and international) of a novel contractual relationships with although the restriction of all variola smallpox antiviral program will require drug companies (Recommendation 1,

11190 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al. below); (ii) invigorating poxvirus re- should engage in research on appro- laboratory facilities. The research cen- search in academic and government priate animal models for poxvirus ters of excellence being established by laboratories (Recommendations 2, 3, infections and on the kinds of studies NIAID’s Biodefense Program can pro- and 4, below); and (iii) forging consen- needed to show efficacy of antiviral vide mechanisms for responding to some sus on the criteria for safety and effec- drugs. The FDA itself also should of these needs. tiveness of bioweapons countermea- receive sufficient funding to support 5. WHO restrictions. NIH and CDC should sures and on the regulations that will its own work on these issues, so that encourage WHO to reevaluate its prohi- govern their development (Recommen- the criteria for licensing smallpox an- bition of certain manipulations of vari- dations 5, 6, and 7, below). tivirals can be established as early as ola virus that would greatly facilitate possible. novel screening methods, for example, Recommendations the introduction of reporter genes into 2. Focused, short-term basic research. 1. Pharmaceutical and biotechnology company Rapid variola virus and the use of variola virus engagement. progress toward deeper understanding genes in other vectors. Mechanisms of poxvirus biology will result from ac- should be developed for undertaking a. We propose an immediate focus on tive collaborations between academic such studies in CDC poxvirus research currently identifiable therapeutic tar- laboratories already engaged in poxvirus laboratories. CDC also should explore gets that are essential enzymes of research and those with specific biologic with WHO whether other qualified lab- poxviruses (see Table 3). To gain the or technologic expertise. NIH should oratories under its control might be participation of major companies, we support established investigators, nation- deemed part of its site and otherwise suggest the following program: ally and internationally, to work with take measures to ensure the continuing, i. direct contact between senior poxvirologists on well defined, short- vigorous function of its poxvirus re- government official and leaders of term projects, in which technology and search effort. major pharmaceutical and bio- personnel are exchanged, and also on 6. Implementation. In view of the impor- technology companies to initiate longer-term, coinvestigator-initiated col- tance of including expert scientific guid- and encourage participation; laborations. The structural biology of ance throughout the steps recommended ii. targeted solicitation of proposals poxvirus proteins, the cell biology of in this report, a high-level advisory and to carry out the screening of poxvirus infection, and the interaction oversight panel, analogous to the AIDS chemical libraries beginning with between poxviruses and the host im- Vaccine Research Working Group, currently identifiable targets and mune system are three areas in which should be created immediately, report- elaboration of an early lead dis- such collaborations should be encour- ing to the heads of NIH, CDC, and any covery phase, followed by federal aged. Establishing the credibility of ani- other federal agencies involved in this support of preclinical develop- mal models for variola infection is also a effort. Membership on this committee ment in those companies that high priority. should represent experience in drug present the most promising dis- 3. Recruiting and training a new cohort of discovery and development, poxvirus covery-phase data; and young investigators. Long-term progress expertise at the highest level, and inter- iii. support of full clinical develop- will require ‘‘new blood,’’ and research national efforts in smallpox antiviral re- ment of the best preclinical initiatives should be designed with this search. Its first, urgent task will be to candidates through negotiated point in mind. establish a specific timeline for drug dis- guarantees of purchase contingent a. NIH should attract new talent into covery and development, including pri- on meeting predetermined prod- poxvirology by granting 1- to 2-year oritization of currently feasible targets uct specifications. fellowships that permit students and and approaches. A second important b. Successful engagement of industry postdoctoral scientists to move between task will be to work out a blueprint for through the process just outlined will laboratories with complementary ex- an effective international effort (see require: pertise and by awarding additional in- Recommendation 7). i. high-level planning and oversight stitutional training grants in viral 7. International participation. The prospect of the process by the advisory pathogenesis. of an intentional release of the variola panel proposed in Recommenda- b. NIH should support one or more lab- virus is a global concern. Research on tion 6; oratory courses in poxvirus research bioweapon countermeasures therefore ii. new procedures for rapid, insight- at sites such as Cold Spring Harbor, should involve broad international col- ful review, including participation NY, or Woods Hole, MA. laborations. There should be no undue of individuals with extensive expe- c. A high-profile fellowship (NIH restrictions on the participation of non- rience in drug discovery and de- Fellow in Viral Pathogenesis) should U.S. citizens in work at U.S. laborato- velopment; be established to support the most ries, and grant and contract funding iii. addressing the issue of indemnifi- outstanding students during postdoc- should extend, where appropriate for cation, through legislation to limit toral studies and their first few years support of the best science, to laborato- the liability of companies that of faculty research. ries outside the U.S. develop smallpox antivirals under this program; and 4. Centralized resources. NIH, with the col- We extend our gratitude to the individuals iv. consortium arrangements that laboration of CDC, should establish re- who attended the National Academies’ June permit companies to share infor- positories and databanks. Large libraries 15–16, 2003, workshop on ‘‘Transforming Bi- mation and thereby reduce risks. of chemical compounds will be particu- ological Information into New Therapies: c. Therapeutics against smallpox will larly useful for academic investigators Smallpox Antivirals,’’ and acknowledge need to be licensed under the FDA’s who have the experience and facilities to NIAID for its support of this activity. In ad- dition, we thank A. Mahmoud, D. Bloom, Animal Efficacy Rule. To promote undertake large-scale screening. NIH and E. Cordes for their useful insights on the sound application of this rule, NIH, and CDC also should create an accessi- pharmaceutical industry, and T. Caskey, M. the U.S. Army Medical Research In- ble central mechanism for facilitating Collett, J. Henney, N. Nathanson, R. Moyer, stitute of Infectious Diseases, CDC, WHO and CDC approvals and for coor- V. Sato, P. Palese, and P. Traktman for help- and the scientific community at large dinating access to the use of national ful comments on the draft of this article.

Harrison et al. PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11191 1. Institute of Medicine (1999) Assessment of Future 10. Rietdorf, J., Ploubidou, A., Reckmann, I., Holm- 22. Pharmaceutical Research and Manufacturers As- Scientific Needs for Variola Virus, ed. Briere, R. strom, A., Frischknecht, F., Zettl, M., Zimmermann, sociation (2003) Insights: Highlights from the Phar- (Natl. Acad. Press, Washington DC). T. & Way, M. (2001) Nat. Cell Biol. 3, 992–1000. maceutical Industry Profile 2003 (PhRMA, Wash- 2. Lane, H. C., Montagne, J. L. & Fauci, A. S. (2001) 11. van Eijl, H., Hollinshead, M., Rodger, G., Zhang, ington, DC). Nat. Med. 7, 1271–1273. W. H. & Smith, G. L. (2002) J. Gen. Virol. 83, 23. National Institute of Allergy and Infectious Dis- 3. White House (December 13, 2002) Protecting 195–207. eases (November 14, 2002) Biodefense Partner- Americans: Smallpox Vaccination Program, press 12. Griffiths, G., Roos, N., Schleich, S. & Locker, J. K. ships: Vaccines, Adjuvants, Therapeutics, Diag- release, www.whitehouse.gov͞news͞releases͞ (2001) J. Virol. 75, 11056–11070. nostics, and Resources, program announcement 2002͞12͞20021213-1.html. 13. Da Fonseca, F. & Moss, B. (2003) Proc. Natl. PAR-03-025. 4. Flint, S. J., Enquist, L. W., Racaniello, V. R. & Acad. Sci. USA 100, 11291–11296. 24. National Institute of Allergy and Infectious Skalka, A. M. (2003) Principles of Virology: Molec- 14. Broyles, S. S. (2003) J. Gen. Virol. 84, 2293–2303. Diseases (August 15, 2002) Small Business ular Biology, Pathogenesis, and Control of Animal 15. Bell, S. P. & Dutta, A. (2002) Annu. Rev. Biochem. Biodefense Program, program announcement Viruses (ASM Press, Washington DC), 2nd Ed. 71, 333–375. PAS-02-149. 5. Moss, B. (2001) in Fields Virology, eds. Knipe, 16. Sodeik, B. & Krijnse-Locker, J. (2002) Trends 25. Institute of Medicine and National Research D. M. & Howley, P. M. (Lippincott Williams & Microbiol. 10, 15–24. Council (2004) Giving Full Measure to Counter- Wilkins, Philadelphia), pp. 2849–2883. 17. Senkevich, T. G., White, C. L., Koonin, E. V. & measures: Addressing Problems in the DoD Program 6. Krauss, H., Weber, A., Appel, M., Enders, B., Moss, B. (2002) Proc. Natl. Acad. Sci. USA 99, to Develop Medical Countermeasures Against Bio- Isenberg, H., Schiefer, H., Slenczka, W., von Grae- 6667–6672. logical Warfare Agents (Natl. Acad. Press, Wash- venitz, A. & Zahner, H., eds. (2003) Zoonoses: Infectious Diseases Transmissible from Animals to 18. Smith, G. L., Murphy, B. J. & Law, M. (2003) Ann. ington, DC). Humans (ASM Press, Washington, DC), 3rd Ed. Rev. Microbiol. 57, 323–342. 26. National Research Council (2004) Biotechnology Re- 7. Seet, B. T., Jonhston, J. B., Brunetti, C. R., 19. DiMasi, J. A., Hansen, R. W. & Grabowski, H. G. search in an Age of Terrorism: Confronting the ‘‘Dual Barrett, J. W., Everett, H., Cameron, C., Sypula, J., (2003) J. Health Econ. 22, 151–185. Use’’ Dilemma (Natl. Acad. Press, Washington, DC). Nazarian, S. H., Lucas, A. & McFadden, G. (2003) 20. Kestin, S. (December 16, 2001) South Florida 27. De Clercq, E. (2002) Nat. Rev. 11, 13–25. Annu. Rev. Immunol. 21, 377–423. Sun-Sentinel, Section A, pp. 1, 9. 28. Institute of Medicine, Board on Health Care 8. Tolonen, N., Doglio, L., Schleich, S. & Krijnse 21. Lee, J.-W. & McKibbin, W. J. (2004) in Institute of Services, Committee on the Evaluation of Vaccine Locker, J. (2001) Mol. Biol. Cell 12, 2031–2046. Medicine and National Research Council Forum on Purchase Financing in the United States (2003) 9. Risco, C., Rodriguez, J. R., Lopez-Iglesias, C., Microbial Threats: Learning from SARS: Preparing Financing Vaccines in the 21st Century: Assuring Carrascosa, J. L., Esteban M. & Rodriguez, D. for the Next Disease Outbreak–Workshop Summary Access and Availability (Natl. Acad. Press, Wash- (2002) J. Virol. 76, 1839–1855. (Natl. Acad. Press, Washington, DC). ington, DC).

11192 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 Harrison et al.