Live Variola Virus: Considerations for Continuing Research (2009)

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1 Introduction T hroughout history, humankind has been plagued by a number of deadly diseases. Smallpox, perhaps the most devastating of these, has always been greatly feared (Morens et al., 2008). The earliest description of smallpox as a distinct clinical syndrome emerged in fourth- century CE China, but earlier records hint at its presence in Europe before then (Damon, 2006). Its causative agent, variola virus, has adapted in unique ways to its only known host species—humans. After centuries of recurring smallpox epidemics that swept through human populations worldwide, impacted the course of history, and killed more than 500 million people, Edward Jenner reported in 1798 that inocu- lation with related viruses, obtained from either cows or horses, conferred immunity to smallpox (Jenner, 1798). More than 150 years later, with this critical tool in hand, the World Health Organization (WHO) in 1959 embarked on an ambitious plan to control and eventually eradicate the disease. In 1977, the last known naturally occurring case of smallpox was recorded in Somalia, and the following year an accidental laboratory- associated infection became the last known case of the disease. In 1979, a commission of health experts certified that natural transmission of smallpox had ceased, and WHO endorsed the declaration a year later in 1980. This final eradication of smallpox represents a monumental event in the history of medicine and public health, and smallpox remains the only intentionally eradicated disease of humankind (Fenner et al., 1998; Tucker, 2001). Since the eradication of smallpox, the global public health community, acting through the World Health Assembly (WHA), has debated the issue of whether stocks of the live variola virus should be retained. In 1983, the  

10 LIVE VARIOLA VIRUS number of centers authorized to house and perform research with the live virus was limited to two—the Centers for Disease Control and Prevention (CDC) in the United States and the Research Institute for Viral Prepara- tions in Moscow, Russia. In 1994, the Russian stocks were transferred to the State Centre for Research of Virology and Biotechnology (VECTOR) in Novosibirsk (Fenner et al., 1998; Tucker, 2001). In 1996, the WHO ad hoc Committee on Orthopoxvirus Infections recommended final destruction of the live variola virus stocks at both research centers, and WHA subsequently set the termination date for 1999. However, the decision to destroy the virus was postponed that year in light of increasing public health and biosecurity concerns, and was ultimately deferred by WHA to assess the potential for continued scientific needs for the live virus (Smallpox Preservation Advisable, 1999; WHO, 1999). In that same year, the Institute of Medicine (IOM) released a consensus report identifying key areas for further scientific research that required the use of live variola virus (IOM, 1999). The conclusions from that report are presented in Box 1-1. Ten years have passed since the 1999 IOM report was issued, and much has since transpired that is relevant to the question of the utility of research using live variola virus. These developments include advances in science and biotechnology, incidents involving bioterrorism, increased investment in research and development on countermeasures, changes in the regulatory approval process, and the reinstitution of smallpox vaccina- tion among civilian and military populations in the United States. Since 1999, WHO has convened a standing Advisory Committee on Variola Virus Research, which has met annually since 1999 and which monitors the state of research in key areas at the two WHO Collaborating Centers for Smallpox and Other Poxviruses. (The reports of the committee’s meet- ings are available through the WHO website.) This committee reports its findings at the annual meetings of WHA, which is the ultimate decision- making body of WHO; these meetings are attended by delegations from all of the WHO member states. WHA has agreed to revisit the issue of variola virus destruction in 2010. In this context, it is important to re-assess the conclusions of the 1999 IOM report and to review the progress that has been made over the last decade. Overview of scientific needs for live variola virus Despite the successful eradication of smallpox 30 years ago, concerns remain about the potential for its reemergence. While a natural outbreak appears unlikely, the threat of intentional reintroduction or accidental release exists. In addition, the emergence of human disease due to monkey­ pox (another member of the Orthopoxvirus genus), including its 2003 

INTRODUCTION 11 BOX 1-1 Conclusions from the 1999 Institute of Medicine Report In 1998–1999, the IOM convened a committee to evaluate the scientific needs for continued retention of live variola virus. The committee identified one short- term need and six long-term needs. Specifically, the committee drew the following conclusions: • Genomic sequencing and limited study of variola surface proteins derived from geographically dispersed specimens is an essential foundation for important future work. Such research could be carried out now, and could require a delay in the destruction of known stocks, but would not necessitate their indefinite retention. • The most compelling reason for long-term retention of live variola virus stocks is their essential role in the identification and development of antiviral agents for use in anticipation of a large outbreak of smallpox. It must be emphasized that if the search for antiviral agents with activity against live variola virus were to be continued, additional public resources would be needed. • Adequate stocks of smallpox vaccine must be maintained if research is to be conducted on variola virus or if maintenance of a smallpox vaccination program is required. Live variola virus would be necessary if certain approaches to the development of novel types of smallpox vaccine were pursued. • If further development of procedures for the environmental detection of variola virus or for diagnostic purposes were to be pursued, more extensive knowl- edge of the genome variability, predicted protein sequences, virion surface structure, and functionality of variola virus from widely dispersed geographic sources would be needed. • The existence of animal models would greatly assist the development and test- ing of antiviral agents and vaccines, as well as studies of variola pathogenesis. Such a program could be carried out only with live variola virus. • Live or replication-defective variola virus would be needed if studies of variola pathogenesis were to be undertaken to provide information about the response of the human immune system. • Variola virus proteins have potential as reagents in studies of human immunol- ogy. Live variola virus would be needed for this purpose only until sufficient variola isolates had been cloned and sequenced. SOURCE: IOM, 1999, pp. 82–85. introduction into the western hemisphere, highlights the importance of research into prevention of orthopoxvirus infection and disease (Reed et al., 2004; Sale et al., 2006; Rimoin et al., 2007; Dubois and Slifka, 2008). At the same time, tools to control potential smallpox outbreaks remain imperfect. While smallpox vaccines based on cross-protection provided by vaccinia-induced immunity are available and are known to be effective from 

12 LIVE VARIOLA VIRUS extensive historical experience, there are still concerns regarding their safety. An estimated 40 percent of those vaccinated with the original and newer, second-generation vaccinia vaccines experience mild to severe adverse reac- tions (McCurdy et al., 2004). The development of third-­generation vaccines with the potential to have a much improved safety profile is currently under way. Additionally, no U.S. Food and Drug Administration (FDA)-approved therapeutics or validated, rapid, point-of-care diagnostics for smallpox are available. The lack of these tools would be an obstacle in the event of a future outbreak of smallpox or other orthopox. Advances made in the fields of molecular biology and genetics in the past 10 years could provide important tools to improve understanding of the structure of variola virus and the functions of its gene products. Additionally, given variola’s specificity for the human species, studying live variola virus in cultured human cells in vitro—including both cells that are targets for initial infection and spread and specialized cells that mediate the immune response—holds the potential to yield new insights into the antiviral mechanisms of host cells and the biology of the human immune system. These findings could provide valuable information not only in the context of controlling DNA viruses, but also as a means of understanding basic inflammatory pathways that can protect or damage the host. Such observations could be extended in appropriate animal models of variola pathogenesis. Complete chemical resynthesis of the variola genome and subsequent production of viable intact virions is now scientifically plausible and technically feasible. This newly emerging and rapidly evolving capabil- ity has profound implications for both the future threat posed by smallpox and the future development of smallpox countermeasures. Box 1-2 summarizes features of the contemporary context in which the scientific needs for live variola virus must be assessed. Current Status of Variola Virus and Materials Because of the biohazard posed by live variola virus, rigorous precau- tionary measures are essential, including strict regulation of the type of facility that is approved for storing and conducting experiments with the virus. Research with live variola virus must be conducted in laboratories with the highest safety and security rating, designated biosafety level 4 (BSL-4) containment facilities. Although other BSL-4 facilities exist, only the two noted above in the United States and Russia are authorized by WHO to perform research with live variola virus under international agree- ment (WHO, 2008). WHO oversees all scientific research with live variola virus, and to ensure the safety of researchers and the security of the virus stocks, periodi- cally conducts inspections of the authorized research facilities. In addition 

INTRODUCTION 13 BOX 1-2 Contemporary Context for Assessment of the Scientific Needs for Live Variola Virus Smallpox as a bioweapon. Smallpox’s virulence makes it an obvious candidate for use as a bioweapon. Historical anecdotes, while not confirmed, suggest that contaminated materials could be used to spread smallpox in target populations. Both the United States and the Soviet Union have engaged in research aimed at weaponizing smallpox. Monkeypox outbreaks. First recognized in humans in 1970, monkeypox is e ­ ndemic in central Africa. Periodic outbreaks have occurred in the Democratic Republic of Congo, with a case fatality rate of 1–10 percent. In addition, the intro- duction of monkeypox in the United States in 2003 demonstrated the continuing threat of orthopoxvirus outbreaks. Immunologically naïve populations. Routine smallpox vaccination ceased in 1980, and earlier in some countries. Almost half the world’s population is currently immunologically naïve to the disease. At the same time, the rise of diseases such as HIV/AIDS that weaken the immune system, as well as the prevalence of atopic dermatitis, would make resumption of routine vaccination difficult. Lack of proper countermeasures. There are today no licensed therapeutics for the treatment of smallpox, and currently licensed vaccines, while effective, are contraindicated for immunocompromised individuals. Resynthesis of the variola genome. Technological advances have led to new breakthroughs, including the complete sequencing of multiple strains of variola virus. The ability to resynthesize viral genomes is well established and may be possible for variola virus. to handling of the live virus, work with the genomic components of the virus is tightly regulated by WHO. Specifically, laboratories other than CDC and VECTOR cannot possess more than 20 percent of the variola genome at any time (WHO, 1994). From its most recent (November 2008) meeting, the WHO Advi- sory Committee on Variola Virus Research reports that access to the BSL-4 laboratories at CDC and VECTOR remains highly controlled and regulated; security procedures are reviewed by WHO, and in the United States by the U.S. Select Agent Program. CDC has also reported on an expansion of its BSL-4 facilities, with another laboratory scheduled to be operational in 2009. Since November 2006, the long-term inventory of variola virus materials at CDC has remained at 451, and genomes from 

14 LIVE VARIOLA VIRUS 45 of the 70 working stock isolates have been sequenced. Withdrawals have been made to ­support WHO-approved projects. Since 2007, a total of 200 nonviable or duplicate working stocks at VECTOR have been destroyed, reducing its collection of variola stocks to 691 registered vials (WHO, 2008). Regulations and Other Guidance Pertaining to Countermeasures for Smallpox Since the IOM’s 1999 report was issued, a number of regulations and other guidance have been promulgated in the United States to guide and facilitate the development and licensure of additional countermeasures for the diagnosis, prevention, and therapy of bioterrorism threats, including smallpox. The most important and directly relevant of these are summa- rized below: • Approval of Biological Products When Human Efficacy Studies Are Not Ethical or Feasible (21 CFR 601 Subpart H, as well as 21 CFR 314 Subpart I for New Drugs). This rule, known as “the A ­ nimal Rule,” was designed to permit approval of drugs and biologics intended to reduce or prevent serious or life-threatening conditions caused by exposure to biological, chemical, radiological, or nuclear substances when human efficacy studies are not ethical and field trials are not feasible (FDA, 2002a). • FDA Guidance for Industry—Smallpox (Variola) Infection: Devel- oping Drugs for Treatment or Prevention (November 2007). This guidance (FDA, 2007a) outlines the unique challenges of develop- ing safe and effective antiviral agents for the treatment and/or pro- phylaxis of smallpox. These challenges include the exceptionally narrow host range of variola virus, the lack of a previously recog- nized effective therapeutic agent, and the lack of human diseases that can be considered closely analogous to smallpox. • The Project BioShield Act of 2004 (Public Law 108–276). This act establishes a comprehensive Emergency Use Authorization (EUA) program that enables the emergency use of medical products against biological, chemical, radiological, and nuclear attacks, real or potential, for both civilian and military personnel. Under this program, the FDA Commissioner can approve the emergency use of drugs, vaccines, medical devices, and diagnostics not previously approved for a particular purpose (FDA, 2007b). • The Public Readiness and Emergency Preparedness Act of 2005 (Public Law 109–148) provides immunity from liability claims aris- 

INTRODUCTION 15 ing from the administration and use of countermeasures covered under EUA. • The Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (the “Bioterrorism Act,” June 12, 2002) (FDA, 2002b). This act states that the “prompt approval of safe and effective new drugs and other therapies is critical to the improve- ment of the public health.” The European Union—primarily through the European Medicines Agency (EMEA), which is responsible for the scientific evaluation of appli- cations for European marketing authorization (licensure) of medicinal products in the European Community—has also focused on the threat of bioterrorism in accordance with Article 57(q) of Regulation (EC) No. 726/2004. This article states that the EMEA shall, with a view to protection of the public health, compile “scientific information concerning pathogenic agents which might be used in biological warfare, including the existence of vaccines and other medicinal products available to prevent, or to treat, the effects of such agents.” EMEA produced a guidance document in 2002 on the use of available medicinal products for the treatment and prophylaxis of biological agents that might be used as weapons of bioterrorism (European Agency for the Evaluation of Medicinal Products, 2002a). Note for Guid- ance on the Development of Vaccinia-Based Vaccines Against Smallpox applies to the development and manufacture of second-generation vaccinia vaccines produced in embryonated eggs or tissue culture (European Agency for the Evaluation of Medicinal Products, 2002b). Finally, in 2003 WHO updated its Recommendations [formerly known as Requirements] for the Production and Quality Control of Smallpox Vac- cine, which had last been revised in 1965 (WHO, 2004). The document acknowledges that global resumption of the production of smallpox vaccine would benefit from modern approaches to production and control, and that present-day regulatory expectations should be met in the licensing process. In addition, the document encourages the development of contemporary international reference materials as guidance for determining the potency of new vaccines and their immunogenicity in vaccinated individuals. study charge and approach In anticipation of the WHA meeting in 2010, CDC requested that the IOM convene a committee to conduct a study on the continued use of live variola virus stocks for research and public health purposes. The charge to the committee is presented in Box 1-3. To address this charge, the IOM convened a committee of experts from both the United States and abroad. Experts in the field of ­orthopoxvirology 

16 LIVE VARIOLA VIRUS BOX 1-3 Charge to the Committee An ad hoc committee of the Institute of Medicine shall conduct a study on the continued use of live variola virus stocks for research and public health purposes. In follow-on to the IOM’s 1999 report, Assessment of Future Scientific Needs for Live Variola Virus, an IOM committee will perform a comprehensive evaluation of the research and development work recommended in that report and completed to date, and consider what unmet needs still exist that require the use of live variola virus. The conclusions and recommendations will inform policy discussions in the United States and within the world community regarding the continued need to retain the official stocks of live variola virus for research purposes, and would provide a major review of completed, ongoing and planned research activities that should be undertaken. The committee shall specifically consider and offer recommendations perti- nent to the utility of live variola virus in addressing potential unmet requirements including: • Advanced development through licensure and post-licensure of antivirals for use in treatment of variola virus infections. • Advanced development through licensure and post-licensure of new, safe and effective vaccine(s). • Development through licensure and post-licensure of less-reactogenic vaccines. • Development of approved protein-based diagnostics which can be used in field situations or diagnostics which have sources of error distinct from those of nucleic acid-based diagnostics. • Improved pathogenesis data to drive therapeutic discovery. were consulted, as well as those with expertise in vaccine, antiviral, and diagnostic development; public health; biosecurity; federal government regulation; and bioethics. The committee held two open workshops to gather information from experts and researchers in the salient fields. A comprehensive search of the scientific literature published on variola and other poxviruses was undertaken, and key literature was assessed. The committee also made inquiries to WHO, CDC, and VECTOR regarding research undertaken outside of the United States that might not be readily accessible in the scientific literature. It is important to note that the committee was charged with assessing scientific needs that require live variola virus. In evaluating unmet needs, the committee recognized the risks of such research and the critical impor- tance of providing independent oversight and essential resources, including BSL-4 facilities when research with live variola virus is undertaken. Com- 

INTRODUCTION 17 menting on retention or destruction of the live variola virus stocks was not within its scope. organization of the report The first four chapters of this report provide context for the question of the scientific needs for live variola virus. Following this introductory chapter, Chapter 2 presents an overview of smallpox and its surveillance and control. Chapter 3 examines variola virus in the context of poxvirology and variola’s similarities with and differences from other orthopoxviruses. Chapter 4 reviews the state of the art with regard to animal models of the pathogenesis and immunobiology of variola and other poxviruses. Chapters 5 through 9 review variola-related research completed since the 1999 IOM report was issued, with emphasis on the role of the live virus in advancing scientific breakthroughs. These chapters also address any unmet or future needs in applications of the research, in terms of both medical countermeasures and any additional knowledge that could poten- tially be gleaned from studying live variola virus. Research in the following areas is examined in turn: genomic analysis (Chapter 5), development of therapeutics (Chapter 6), development of vaccines (Chapter 7), detection of variola and diagnosis of smallpox (Chapter 8), and scientific discovery (Chapter 9). The final chapter presents the committee’s conclusions and recommendations. REFERENCES Damon, I. K. 2006. Poxviruses. In Fields’ virology, 5th ed., edited by B. N. Fields, D. M. Knipe, P. M. Howley, and D. E. Griffin. Philadelphia, PA: Lippincott Williams & Wilkins. Pp. 2947–2976. Dubois, M. E., and M. Slifka. 2008. Retrospective analysis of monkeypox infection. Emerging Infectious Diseases 14(4):592–599. European Agency for the Evaluation of Medicinal Products. 2002a. EMEA/CPMP guidance document on use of medicinal products for treatment and prophylaxis of biological agents that might be used as weapons of bioterrorism. CPMP/4048/01. http://www.emea. europa.eu/pdfs/human/bioterror/404801.pdf (accessed January 16, 2009). European Agency for the Evaluation of Medicinal Products. 2002b. Note for guidance on the development of vaccinia-based vaccines against smallpox. http://www.emea.europa. eu/pdfs/human/vwp/110002en.pdf (accessed January 26, 2009). FDA (Food and Drug Administration). 2002a. New drug and biological drug products; evi- dence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. Federal Register 67(105). http://www.fda.gov/cber/rules/humeffic. htm (accessed January 13, 2009). FDA. 2002b. The Bioterrorism Act of 2002. http://www.fda.gov/oc/bioterrorism/bioact.html (accessed January 13, 2009). 

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