12
Summary and Conclusions

Smallpox, caused by variola virus, is a devastating disease with high case-fatality and transmission rates. Inoculation with vaccinia virus is highly protective against natural infection with variola virus. Vaccination, together with the restricted host range and vigilant surveillance efforts, enabled a worldwide containment and inoculation program to eliminate smallpox globally more than 20 years ago. The last case of naturally occurring smallpox was in Somalia in 1977. Known tissue collections containing live variola virus material were subsequently consolidated in two international repositories in the United States and Russia.

Scientific research on live variola virus requires maximum containment facilities. As a consequence, little research on variola has been done since eradication. During that same period, scientific knowledge about the molecular pathogenesis of many viral infections has become considerably more sophisticated through studies of the immunology, virology, molecular genetics, structural biology, and molecular pharmacology of infection. While such investigations enable effective diagnosis, treatment, or prevention of many other viral infections, increased knowledge of variola infection has been limited largely to the cloning and complete sequencing of two strains of variola major from the Asian subcontinent, partial sequencing of one strain of variola major, and one strain of variola minor from Latin America. In addition, a few genes of other strains have been sequenced.

Since the eradication of smallpox, virologists have come to realize that disease-causing viruses are efficient pathogens because of a broad spectrum of mechanisms that can defeat or alter innate defenses or immune system responses. The technologies that have been developed to investigate these phe-



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--> 12 Summary and Conclusions Smallpox, caused by variola virus, is a devastating disease with high case-fatality and transmission rates. Inoculation with vaccinia virus is highly protective against natural infection with variola virus. Vaccination, together with the restricted host range and vigilant surveillance efforts, enabled a worldwide containment and inoculation program to eliminate smallpox globally more than 20 years ago. The last case of naturally occurring smallpox was in Somalia in 1977. Known tissue collections containing live variola virus material were subsequently consolidated in two international repositories in the United States and Russia. Scientific research on live variola virus requires maximum containment facilities. As a consequence, little research on variola has been done since eradication. During that same period, scientific knowledge about the molecular pathogenesis of many viral infections has become considerably more sophisticated through studies of the immunology, virology, molecular genetics, structural biology, and molecular pharmacology of infection. While such investigations enable effective diagnosis, treatment, or prevention of many other viral infections, increased knowledge of variola infection has been limited largely to the cloning and complete sequencing of two strains of variola major from the Asian subcontinent, partial sequencing of one strain of variola major, and one strain of variola minor from Latin America. In addition, a few genes of other strains have been sequenced. Since the eradication of smallpox, virologists have come to realize that disease-causing viruses are efficient pathogens because of a broad spectrum of mechanisms that can defeat or alter innate defenses or immune system responses. The technologies that have been developed to investigate these phe-

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--> nomena have advanced dramatically in the past 20 years and will almost certainly become even more powerful in the future. As a consequence of these capabilities, novel approaches to biomedical research have emerged. Techniques have been developed to render viruses safer to use in laboratory studies and to provide new animal models with which such studies can be performed. Because variola virus is the only uniquely human orthopoxvirus, it offers the potential for understanding aspects of human biology that may have considerable biomedical significance. Thus variola virus, once considered an agent of human pestilence, may in the future be viewed as a potential source of knowledge and of reagents to support advances in cell biology and immunology. In particular, research using variola virus could assist in understanding the inflammatory response, which is a key process of cell-mediated defense. In preparation for international deliberations concerning whether all variola virus stocks, stored clinical materials containing variola virus, and live variola virus genome DNA held in the international repositories are to be destroyed, this committee was asked to assess future scientific needs for live variola virus. The committee was not asked to make a recommendation about destruction or retention of variola virus stocks, and such a determination involves information beyond the purview of the committee. The Broader Context In carrying out its charge, the committee recognized that the knowledge likely to be obtained from future research using live variola virus must be assessed within a broader context that has changed dramatically since the eradication of smallpox. This broader context encompasses three major global conditions. First, since the cessation of vaccination programs following the successful eradication of smallpox, the entire global population has become more susceptible to the disease than ever before. Widespread inoculation during the eradication program produced a high level of immunity within the general population that protected those exposed to the virus. A smallpox outbreak occurring today in a highly mobile and susceptible population, in contrast, might spread widely before being recognized and before effective countermeasures could be put in place. If large enough, an outbreak could quickly overwhelm medical response capabilities. Second, the significant number of individuals in many parts of the world who are immunocompromised as a result of HIV infection, an organ transplant, or chemotherapy limits the potential widespread use of the current smallpox vaccine because it is made from live vaccinia virus. Smallpox vaccination with the vaceinia vaccine, the workhorse of the eradication program, was terminated primarily because of eradication, but also because of concern about rare but

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--> serious medical complications that occurred among children with highly compromised cellular immune systems or severe dermatitis. Such problems could be expected to be more prevalent in populations with substantial proportions of immunocompromised individuals. Third, variola virus is increasingly considered a serious threat as a biological weapon. There is currently concern that the development, production, and stockpiling of weapons based on viruses, bacteria, and fungi have continued. Testimony before a committee of the U.S. Congress, for example, alleged that scientists in the former Soviet Union continued to experiment with these materials on a large scale despite such experiments being outlawed by the Biological Weapons Convention in 1972. Although known stocks of variola virus have been consolidated in two repositories, human tissue and clinical laboratory materials infected with variola virus were widespread before eradication. The high human-to-human transmission rates of smallpox, its devastating medical consequences, and the difficulty of mounting countermeasures all contribute to the attractiveness of variola virus as a terrorist weapon. The probability that variola could reemerge as a threat because of unregistered growth of clandestine virus is an as yet unquantifiable parameter in estimation of the scientific utility of retaining variola virus. Live variola virus would be required for full development of antiviral therapeutics needed to deal with such a threat. Scientific Needs for Live Variola Virus The committee's charge was restricted to assessment of scientific needs for live variola virus. It did not include consideration of risks that may be associated with retention of the existing stocks, and no attempt was made to determine whether the scientific needs identified by the committee outweigh these risks. Furthermore, the committee did not address the likelihood that the funds and other resources needed to pursue this research, including facilities with suitable biological containment provisions, would be available. It must also be recognized that predicting the future is impossible, and while the committee has done its best to provide an assessment of future scientific needs for live variola virus, the unfolding of actual needs and opportunities is likely to depend on the emergence of unforeseeable technical developments, experimental tools, and model systems. For these reasons, the committee expresses its findings and conclusions below in conditional form: If particular knowledge or capability were to be pursued, would the associated research require live variola virus? The committee identified six potential areas of research that could require the use of variola virus, and then evaluated for each area whether live virus would be needed for that purpose. Before addressing these six areas, however, the committee notes a need associated with the short-term use of variola virus stocks.

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--> 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. We turn now to the six areas of research examined by the committee with regard to the potential need for live variola virus. 1. 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. Live variola stocks would have to be maintained if the development of effective antiviral drugs for smallpox therapy and prophylaxis were to be pursued. There is currently no effective antiviral for the treatment or prevention of smallpox. Vaccination, which reduces the severity of the disease if administered within 4 days of exposure, is currently the only recourse for those infected with the disease. Moreover, vaccine supplies have dwindled and may be deteriorating. In addition, as noted earlier, vaccinia vaccine, which is used for smallpox immunization, is a live virus and cannot be used safely with immunocompromised individuals. Having a number of antiviral agents would provide greater protection against an emergence of drug-resistant variola virus, whether the result of natural evolution or genetic engineering. If new agents were to be developed, cell culture infection assays would be important for demonstrating their activity and effectiveness, and for determining the concentration required to prevent infection or its spread. Some of this testing could be carried out with replication-defective forms of variola virus cultured in cells engineered to complement the defect in the virus. Such replication-defective forms of vaccinia virus have been constructed. Yet other steps in this testing would require the use of live variola virus and recently isolated human cells since measurement of tissue culture activity using other orthopoxviruses or replication-defective forms of variola virus and genetically engineered cell lines could yield misleading results. Finally, private enterprise has little incentive to undertake the development and testing of agents for smallpox prevention and prophylaxis. Therefore, such studies would be dependent on the availability of public resources. 2. Adequate stocks of smallpox vaccine would have to be maintained if research were to be conducted on variola virus or if main-

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--> tenance of a smallpox vaccination program were required. Live variola virus would be necessary if certain approaches to the development of novel types of smallpox vaccine were to be pursued. Vaccinia virus vaccine was effective in eradicating smallpox. As noted earlier, however, current stocks of vaccinia vaccine are limited and may be deteriorating. If it again became necessary to control smallpox with a vaccination program, the current supply would need to be replenished. In addition, if laboratory research using variola virus were to be continued, vaccine would have to be available for laboratory workers, even in the absence of an outbreak. Retention of live variola virus for vaccine production would not be required if vaccinia vaccine supplies were replenished using established methods of manufacture. Moreover, production of vaccinia vaccines using tissue culture could be pursued without the use of live variola virus. Vaccines derived from tissue culture could be compared with the standard vaccine by evaluation of reactogenicity and immunogenicity (essentially the ''take" and immune potency of the vaccine) in human subjects and by laboratory assays. Live variola virus would be required only for testing of novel vaccine development strategies using materials other than live vaccinia virus, such as a DNA vaccine expressing selected variola genes. The above-noted concern about the safety of using vaccinia virus vaccine in populations with high levels of HIV infection or other immunosuppressive conditions is the reason for developing nonstandard vaccines. Since definitive evidence of the protective efficacy of such vaccines could not be obtained in the absence of an outbreak, laboratory testing using live variola virus in as yet undeveloped animal models would be needed for this purpose. 3. If further development of procedures for the environmental detection of variola virus or for diagnostic purposes were to be pursued, more extensive knowledge of the genome variability, predicted protein sequences, virion surface structure, and functionality of variola virus from widely dispersed geographic sources would be needed. Evaluation of the specificity and sensitivity of detection methods for variola virus and other orthopoxviruses would require increased knowledge regarding the DNA sequence not only of variola virus from multiple geographic locations, but also of other orthopoxviruses, especially monkeypox. Development of detection and diagnostic procedures would require field or experimental animal model testing. Such efforts might be carried out using a vaccinia virus with one or more variola virus genes instead of live virus. Although such approaches would be preferable to the use of live virus, current policies prohibit the production and use of recombinant vaccinia virus containing a variola virus gene(s). Moreover, use of recombinants is not likely to fully resolve sensitivity

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--> and specificity requirements, because the fidelity of function and level of expression of engineered recombinants may be different from those of variola virus itself. Limited studies with monkeypox virus and the homologous monkeypox protein might be undertaken, however, to confirm the recombinant vaccinia virus data. An adequate database of the abundance and molecular interactions of variola virus surface proteins would enable reasonable comparisons. Despite residual uncertainty as to whether a surface protein-based detection strategy would work for variola once a sufficient number of variola genomes had been cloned and sequenced and their surface proteins analyzed, the use of live variola virus would not add information worth the risk of exposure to live virus. It may be possible to obtain insight into variations by restriction fragment length polymorphism comparison of variola genes amplified by polymerase chain reaction, but the precise nature of individual gene variation and resultant impact on the protein product(s) requires more detailed sequencing. As noted earlier, sequencing of more variola virus isolates might require a delay in the destruction of known stocks of live virus, but would not necessitate theft indefinite retention. 4. The existence of animal models would greatly assist the development and testing of antiviral agents and vaccines, as well as studies of variola pathogenesis. Such a program could be carried out only with live variola virus. The major rationale for testing agents in animal models is to reduce the risk to human subjects and to optimize the design of clinical trials. Given that there is no opportunity to assess the efficacy of agents in infected human subjects short of an outbreak, there would be a serious need for animal model studies if new antiviral agents were to be developed. In the event of an outbreak, moreover, clinical conditions would not lend themselves to rigorous testing of experimental agents. The current absence of suitable animal models for variola virus does not mean that such models could not be developed in the future, given advances in reconstituting certain experimental animals with human genes and cells derived from humans. Virus that had been genetically modified to be defective in its replication could be introduced into animals capable of expressing the needed genes. These and other advances could make testing in animals feasible and safe, and provide protection against a variola virus outbreak. 5. 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. The specific spatial and temporal patterns of variola virus gene expression must be deciphered in the context of infection at the level of cells, organs, and

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--> animal models. Studies of these phenomena could provide information on how the virus manipulates the human immune response in order to spread, on the mechanisms of cell death, and on numerous other aspects of variola infection. 6. Variola virus proteins have potential as reagents in studies of human immunology. Live variola virus would be needed for this purpose only until sufficient variola isolates had been cloned and sequenced. Virus-encoded proteins that function as immunomodulators are particularly abundant in poxviruses, and variola virus is uniquely adapted to the human immune system. Thus, it is possible that variola virus could serve as a resource for the discovery of human-specific reagents, including such diverse examples as cytokine inhibitors, anti-inflammatory proteins, and regulators of apoptosis. Finally, the future scientific needs for live variola virus must be assessed in light of the knowledge that might be derived from studies of other orthopoxviruses, variola virus DNA clones, orthopoxvirus with one or more variola genes, replication-defective variola virus, live variola virus in tissue culture, and live variola virus in animal models. Table 12-1 summarizes this comparison and provides references to more extensive discussion in earlier chapters. Overall Conclusions The most compelling need for long-term retention of live variola virus is for the development of antiviral agents or novel vaccines to protect against a reemergence of smallpox due to accidental or intentional release of variola virus. In addition, much scientific information, particularly concerning the human immune system, could be learned through experimentation with live variola virus. Indeed, the weight of scientific opinion suggests that continuing investigation of variola virus could lead to new and important discoveries with real potential for improving human health. At the same time, the committee recognizes that limited research infrastructure and resources constrain the realization of this potential. Fortunately, recombinant DNA technology has progressed such that it is now possible to render variola virus incapable of replicating or causing disease. Such genetically crippled variola virus could be used for some steps in the testing of antiviral agents and for some scientific studies. The risks of retaining the stocks of live variola virus might well outweigh the benefits. If the stocks were retained, however, they could offer the possibility of scientific advances that could not otherwise be achieved.

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--> Table 12-1 Potential Scientific Needs for Poxviruses and Their Components Research Area Other Orthopoxvirus Variola Vires DNA Clones Orthopoxviruses with One or More Variola Genes Replication-Defective Variola Virus Live Variola Virus in Tissue Culture Live Variola Virus Animal Models* Variability of Variola (Chapters 8, 9) Useful for comparison Essential Helpful Not required Essential until overlapping and repetitive clones from many isolates are available Not required Antivirals (Chapter 6) Helpful Helpful Helpful Helpful Essential for development steps Essential for full verification Tissue Culture Vaccines (Chapter 7) Essential No use Helpful Possible verification Not required Essential for full verification Novel Vaccines (Chapter 7) Helpful May be used to develop immunogens Not applicable Helpful Helpful for development Essential for full verification Environmental Detection (Chapter 8) Essential for early phases Essential for assessing variola variability Helpful for field testing Partial verification Helpful but not essential Helpful but not essential Diagnosis of Infection (Chapter 8) Helpful for determining specificity Essential Essential for in vitro or in vivo testing Helpful but not essential Helpful but not essential Helpful but not essential Virus-Cell Interactions (Chapter 10) Helpful Helpful Helpful Helpful Essential for some targets Helpful but not essential Virus-Host Interactions (Chapter 10) Helpful Helpful Helpful Helpful Helpful for some targets Essential for some targets * Currently unavailable.