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s Medical Intervention and Technological Solutions OVERVIEW The post-eradication era is a period of history for which there has been no precedent whatsoever in terms of a zero base of immunity. Cessation of immunization will eventually create a population susceptible to widespread infection in the event of accidental or intentional reintroduction or re- emergence of the eradicated virus. Thus, even after immunization ceases, vaccine production must continue. However, many currently available vaccines may not be appropriate for continued post-eradication vaccine production or reinstatement. Vac- cines must be continually improved and ongoing vaccination research main- tained. Other potentially useful antiviral strategies antivirals, prophylaxis, and probiotics must also be considered as means to strengthen the im- mune system and serve as adjuvant or prophylactic therapies. In the case of polio, for example, it remains to be determined which vaccine (oral polio vaccine [OPV] or inactivated polio vaccine [IPV]), or variant thereof, should be produced in the post-eradication, post-vaccina- tion era. A detailed plan for vaccine production will require more informa- tion on OPV-derived viral persistence and transmission, as well as continu- ing dialogue between public health and research communities in order to ensure that appropriate vaccination research continues. The immune system may face unforeseeable challenges when immunity in the community at large wanes in the post-immunization era, and even immunized individuals may be at risk. Molecular biology technology has 121

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122 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION advanced to the point where antiviral drugs couici be developed to target specific viruses. With the exception of HIV and influenza, however, ctis- eases for which antiviral therapy has been consiclered are not usually con- sidered epidemic. The research community and pharmaceutical industry must make a concerted commitment to developing antiviral therapies for use as potential adjuvants for vaccine-preventable diseases. Immunoprophylaxis includes both nonspecific approaches to stimula- tion of innate antiviral defenses and specific prophylaxis clirected at par- ticular pathogens. Currently, the best understood nonspecific prophylaxis is interferon (IFN) A/. However, viruses display tremendous variability in their reponses to the effects of IFN alp, and many viruses have evolved ways around IFN a/,B's antiviral pathways. As is the case for antivirals, technology has advanced to a point where specific prophylactics could be developed for use against vaccine-preventable diseases including small- pox, polio, and measles but this has not been done. Finally, current studies suggest that probiotic bacteria living microbes introduced into the body in order to improve intestinal microbial balance- could be used to strengthen the immune system, even in immuncompromised individuals. Novel microbial mechanisms need to be further studied for their potential use as antigen delivery vehicles and adjuvants. In an age of unprecedented successful vaccination initiatives, public and private sector support has led to the rapid development of vaccines for numerous infectious diseases. Implementation of these products has helped encourage confidence in the biomedical research and public health commu- nities and garnered political will for disease eradication initiatives. This support, confidence, and political will must continue in the post-eradica- tion era. Strong commitment is needed from both the public and private sectors to share the costs and risks associated with developing new vaccines and therapeutic products which may have only a very short product life cycle. Effective and appropriate antiviral therapies are critical for the pro- tection of future populations in a post-immunization era. THE POLIO ERADICATION EFFORT: SHOULD VACCINE ERADICATION BE NEXT; Vincent R. Racaniello, Ph.D. Higgins Professor, Department of Microbiology Columbia University College of Physicians and Surgeons New York, NY The World Health Organization (WHO) goal to eradicate polio by the year 2000 (now extended to 2005) has resulted in an extraordinary recluc-

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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 123 tion in global incidence of the disease. According to the WHO global plan, vaccination can stop when eradication is certified, laboratory stocks of poliovirus are contained, and there is no evidence of persistent vaccine- derived poliovirus circulation (WorId Health Assembly, 1988~. Although eradication may eventually be certified, it is likely that poliovirus will never be completely contained, and recent findings indicate that vaccine-derived polioviruses can circulate and cause disease. Consequently, vaccination will probably not be discontinued anytime in the foreseeable future. Although the use of live, attenuated polio vaccine (OPV) has been crucial to the success of the eradication program thus far, unique properties of the vaccine complicate the decision to cease vaccination. Before we can stop vaccination, we must answer the following questions: 1. How long will OPV persist after it is no longer administered to humans? Will such persistence (including virus excreted by immunocom- promised individuals) constitute a threat to the growing number of unvac- cinated individuals? 2 What is the transmissibility of OPV strains? 3. Will it be possible to eliminate all potential sources of poliovirus in the post-vaccine era? 4. How will we respond to an outbreak of polio in the post-vaccine era? 3t Poliovirus infections, which are transmitted by fecal-oral contamina- tion, begin in the pharyngeal and intestinal mucosa before spreading to the blood and invading the central nervous system. Paralytic disease, which occurs in about 1 in 100 infections, results from the destruction of motor neurons. Poliomyelitis can be prevented by the use of either an injected, formalin-inactivated vaccine (inactivated polio vaccine, IPV), or a live, at- tenuated vaccine which is taken orally and replicates in the intestine (oral polio vaccine, OPV). Both vaccines generate humoral immunity, but only OPV produces local antibodies in the intestine. As a result, wild poliovirus can replicate in the gut of individuals immunized with IPV, but not in the gut of those immunized with OPV. The OPV strains used in the WHO eradication effort were developed by Albert Sabin, who identified variants of the three poliovirus serotypes that were immunogenic but did not cause disease. Since then, molecular biological tools have been used to identify the mutations responsible for the attenuation phenotypes of the vaccine strains. In the 1980s, scientists dis- covered that these mutations revert to pathogenicity during replication in the human gut, which explains why OPV-shed virus is more neurovirulent than the administered parent virus. Will virulent, OPV-derived viruses shed by vaccinees be a threat in the

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124 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION post-vaccination era? To answer this question, we must first consider how long these viruses persist in the environment. In a recent stucly carried out in Japan, neurovirulent, OPV-derived viruses were isolated from sewage and river water up to three months after routine immunization (Yoshida et al., 2000~. The authors concluded that there is an environmental risk of vac- cine-associated polio as long as live vaccine is not replaced by inactivated vaccine. Similar studies in Cuba suggest that OPV may persist in the popu- {ation for several months after vaccination. During the type 3 polio epi- demic in Finland in 1984, OPV was detected up to six months after mass immunization. All of these studies were conducted in communities with a high proportion of immune individuals; it is not known if the level of immunity to poliovirus affects the duration of persistence. The problem of OPV persistence is further complicated by the observa- tion that immunocompromised individuals who receive OPV may excrete virus for extended periods. For example, in one study, a patient who re- ceived monotypic Sabin type 3 in 1962 excreted neurovirulent type 3 virus for 637 days with no symptoms of polio (Martin et al., 20001. Individuals with B cell deficiencies often go undiagnosed and may excrete enteroviruses for long periods. The extent to which immunodeficient individuals are infected with polio is unknown and needs to be determined. After the cessation of polio immunization, OPV will likely continue to circulate in most populations for at least a few months, perhaps up to a year. At the same time, the number of susceptible individuals will increase. This raises the questions: will OPV-derived viruses pose a threat to unvac- cinated indivicluals, and can OPV-derived viruses be transmitted and cause disease in humans; As long as OPV has been in use, scientists have recognized its transmis- sibility among humans. Numerous studies have documented the develop- ment of anti-poliovirus antibodies in nonimmunized persons in communi- ties undergoing vaccination. For example, in one study of a U.S. Amish community where many individuals refuse vaccination, 89% of unvacci- nated children developed antibodies to type 2 poliovirus, presumably from circulation of the vaccine virus from neighboring areas where the vaccine was usecI. This ability to immunize non-vaccinated individuals is considered to be an advantage of OPV, especially in Third World countries where immunization levels are low and poor sanitation promotes extensive virus spread. However, in the post-eraclication era, live vaccine strain transmissi- bility will be a liability. It will be ironic if it becomes necessary to continue vaccination as protection against vaccine-derived polioviruses. Several recent studies confirm that OPV-like strains excreted after im- munization can be transmitted and cause poliomyelitis among humans. In 2000, a neurovirulent derivative of the Sabin type 2 OPV strain was iso-

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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 125 lated from sewage in Israel (Shulman et al., 2000~. The extent of sequence divergence of this strain from Sabin type 2 indicates that it had probably been replicating in one or more people for at least six years. These observa- tions indicate that OPV-like virus can be transmitted "silently," i.e., in the absence of disease, in an immunized population. In Egypt, 32 polio cases that occurred from 1988-1993 have been attributed to a type 2 vaccine- derived poliovirus strain (Centers for Disease Control and Prevention [CDCI, 2001~. Analysis of the virus isolate sequences indicates that they were probably derived from a single infection in 1982, the progeny of which circulated in Egypt for the next 10 years. During Tuly and November 2000, an outbreak of poliomyelitis occurred in Hispaniola (CDC, 20003. The virus responsible for this outbreak was derives! from the Sabin type 1 strain. Sequence analysis indicates that it had been circulating in the region for approximately two years. All of these findings demonstrate that neurovirulent revertants of OPV can be transmitted among humans and cause poliomyelitis. In light of this information, it is impossible at this time . . , . . . . . to plan cessation ot 1mmumzatlon against po 1O. In order to prevent reintroduction of the virus in the post-vaccination era, a crucial component of the eradication effort is the identification and destruction of poliovirus stocks. It will be an enormous task to track clown every poliovirus stock, particularly in light of the absence of an enforce- ment authority. We cannot simply depend on the good will of nations, as suggested by WHO. An even greater challenge is identifying clinical labora- tories that unknowingly harbor poliovirus. Finally, how do we deal with a situation in which, for example, a tube labelecl "Coxsackievirus B3" actu- ally contains poliovirus type 2? Since this has actually occurred, it is not a hypothetical threat but a real possibility. A paradox that arises in the post-OPV era is that it will be critically important to continue producing vaccine stocks for use in the event of a disease outbreak. In populations that have lost immunity to the virus, a poliovirus vaccine production facility will be a hazard equivalent to a bioweapons plant. With smallpox, this problem was avoided because of the strain differences between the vaccine and wild viruses, but poliovirus vac- cines do not offer such an easy solution. Which poliovirus vaccine will be produced in the post-OPV era? Be- cause the inactivated polio vaccine (IPV) is produced from wild-type strains of poliovirus, its production would require a high containment facility. Alternatively, IPV might be produced from the Sabin poliovirus strains, although some research would be required to demonstrate the feasibility of this approach. However, immunization with IPV would not prevent intesti- nal carriage of the virus, increasing the likelihood of spread of the virus in the population. Vaccination with OPV would probably be more effective in

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126 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION curtailing epidemics of poliomyelitis, but excretion of vaccine-derived OPV would be problematic for reasons discussed above. There are no easy answers to these questions, but it is disturbing that a cletailed plan for poliovirus vaccine production in the post-OPV era has not been formulated. Failure to present a coherent plan for the production of vaccine stocks in the post-vaccination world is another reason why we . . cannot stop vaccinating. The plan to eradicate polio has had an unfortunate effect on poliovirus research. As noted recently in an article entitled "Don't Underestimate the Enemy" in Nature, January 18, 2001, "When an infectious disease appears to be in decline the agent that causes it tends to disappear from the biomedi- cal research agenda." In the late 1990s, WHO and CDC began informing polio research laboratories that they would soon be required to cease polio- virus research and destroy virus and infectious DNA stocks. Although the exact date was somewhat vague, the resulting uncertainty inhibited poliovi- rus research. Graduate students and postdoctoral fellows no longer viewed working on poliovirus as a wise career option, and funcling agencies and their peer review groups began to question the wisdom of long-term (five- year) investment in research programs on the virus. This effect was unfortu- nate, because many projects relevant to the eradication effortwork on new vaccines, animal models for virus transmission, and anti-viral com- pounds (which might be useful in a post-vaccination-era outbreak of po- lio) did not proceed. WHO decided not to continue poliovirus research in 1988 because the virus would be eradicated by 2000! Today it is quite clear to many virologists that it might not be possible to eliminate poliovirus from the world. It therefore seems unfortunate that the poliovirus research establishment has been substantially depleted, espe- cially since questions relevant to the eradication effort have not been ad- equately addressed. One of the lessons we have learned from the polio eradication effort is that there continues to be a large gap between basic research and public health. For example, the research community has doubted whether it will be possible to eliminate poliovirus ever since the eradication goal was first announced in 1988. Nevertheless, the force of public health policy has overriden these concerns, resulting in the disman- tling of research programs that could otherwise have contributed to the eradication effort. Future eradication campaigns should benefit from this experience. Although it is important to convince governments and health authorities that a disease can be eradicated, it is also important to maintain communication with the research community so that crucial research con- t~nues.

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MEDICA ~ INTER VENTION AND TE CHNOL O GICAL SOL UTIONS 1 2 7 ANTIVIRAL THERAPY IN THE MANAGEMENT OF POST- ERADICATION INFECTIOUS DISEASE OUTBREAKS Richard f. Whitiey, M.D. Loeb Eminent Scholar Chair and Professor, Department of Pediatrics University of Alabama at Birmingham, Birmingham, AL Prevention must take precedence over treatment of infectious diseases. In an age of unparalleled successful vaccination, particularly when the eradication of smallpox has been documented and the eradication of polio- virus is anticipated, one must question the necessity of developing antiviral drugs targeting infectious diseases slated for global eradication. Successful immunization against measles, mumps, rubella, diphtheria, and many other pathogens has been demonstrated worldwide, though with varying degrees of success. There are several different circumstances under which re-emergence of an infectious agent might be anticipated: Bioterrorism (e.g., deployment of smalIpox), Resurgence of an infection thought to be eradicated (e.g., poliomy- elitis in Santo Domingo), and Clinical reactivation of a vaccine-preventable latent virus (e.g., va- ricella) transmitted to a high-risk susceptible (seronegative) individual. This overview focuses on the potential utility of specific antiviral and more generalized broad-spectrum antiviral agents in a post-eradication vac- cine era. Available Therapeutic Resources The armamentarium of the public health physician with regard to anti- viral agents is limited, at best. Successful antiviral therapy has only been demonstrated in four general infectious areas: 1. The management of influenza virus infections with tricyclic amines and neuraminidase inhibitors, 2. The treatment of HIV infection with reverse transcriptase inhibi- tors, protease inhibitors, and other novel therapeutics, 3. The therapy of several herpes virus infections, including herpes simplex virus, cytomegalovirus, and varicella zoster virus, with nucleoside and nucleotide analogs, and 4. Therapeutic interventions for hepatitis B and hepatitis C with nucleoside analogs and interferons.

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128 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION While therapy for each of these broact infectious disease agents has been shown to be clinically efficacious, resulting in decreased morbidity and mortality, no therapeutic intervention should supplant disease preven- tion by vaccination. Toward this end, some therapeutic agents have been cleveloped for pre-emptive antiviral therapy to be administered before overt disease but in the presence of viral antigenemia. This approach has proven very successful in the management of cytomegalovirus disease in organ . . transplant recipients. Currently, none of the immunological interventions or modulators (e.g., interferon or interferon-like compounds) have proven valuable in the pre- vention of viral disease. With the exception of limited monoclonal antibod- ies (e.g., palivizumab for respiratory syncytial viruses), disease prevention has not been achieved by this modality. Public Health Implications With the exception of influenza and HIV infections, those diseases for which antiviral therapy exists are not usually considered epidemic. Diseases that could take on epidemic proportions namely, smallpox, measles, ru- bella, polio, dengue, ant! Ebola have never been considered candidates for antiviral drug development. This is alarming in light of the fact that extrem- ist governments or individuals will likely consider using these agents as bioterrorist weapons in the post-vaccine eradication era when seroprotec- tion will have waned in the community at large. Scientists have identified molecular targets amenable to the develop- ment of selective and specific antiviral agents. The knowledge of viral-host interactions should lead to the development of specific and more general- ized modulators of host response, such as induction of intracellular inter- feron pathways. The unique properties of each virus need to be considered when devel- oping selective, specific inhibitors to viral replication. For example, several viruses primarily the herpes viruses but also hepatitis B and C- have a propensity to establish latency. Recognizing that reactivation can occur, even with an effective vaccine, exposure of susceptible (seronegative) or non-vaccinated individuals could result in exaggerated disease. An example of this is the reactivation of varicelIa poster virus which results in shingles, or chickenpox. Shingles is contagious for seronegative individuals and is always more severe in adults than in children. Changes in the antigenic nature of an organism may also render it more pathogenic for the population at large. This phenomenon has Greatly been documented by the detection of the.HSN1 influenza A strain in Hong Kong. It is anticipated that a major antigenic shift in the near future will result in a worldwide influenza pandemic. The lack of adequate vaccine

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MEDICAL INTERVENTION AND TECHNO LOGICAL SOLUTIONS 129 stores and vaccines containing the appropriate antigens, combined with the inability to generate sufficient quantities of antiviral drugs, would leave the worId's population at significant risk for disease caused by pandemic influ- enza. Conclusion In an era of rapid vaccine deployment and committed attempts at worldwide eradication of diseases other than smallpox, questions regarding the need to develop additional antiviral agents are very serious. With the lingering threat of bioterrorism, the availability of therapeutics to treat vaccine-preventable diseases, such as smallpox, should be considered a high . . prlorlty. It is impossible to envision a universal vaccine program for the preven- tion of such diseases as rabies, Ebola, dengue, and others. However, all of these viruses are amenable to the development of specific antiviral agents. Molecular biology tools are now available for the development of anti- viral agents. Plus, the knowledge derived from developing therapeutics for one virus can be applied to other viruses. For example, therapeutics di- rected against polio can be applied to other members of the Picornavirus family, including hepatitis A virus, rhinoviruses, enteroviruses, and coxsackieviruses, all of which cause significant morbidity in the worId's population. Toward this end, the pharmaceutical industry must make a commitment to the development of antiviral interventions. POTENTIAL USE OF CYTOEC[NES AND ANTIBODY FOR POST- EXPOS~E PROPHYLAXIS IN THE POST-ERADICATION ERA Diane E. Griffin, M.D., Ph.D. Professor and Chair, W. Harry Feinstone Department of Molecular Microbiology and Immunology Johns Hopkins School of Hygiene and Public Health, Baltimore, MD An important benefit of viral eradication, in addition to elimination of morbidity and mortality due to infection, is the elimination of the need for continued immunization of large numbers of people. Discontinuation of universal immunization will result in considerable cost savings. However, it will also eventually create a population susceptible to widespread infection in the event of reintroduction or re-emergence of the eradicated virus. Because reintroduction will always be a possibility, even in the best-con- trollecl circumstances, it is necessary to have a planned response if and when it should occur. There are several possible responses:

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130 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION Resumption of widespread immunization, assuming that the vac- cine and vaccine-manufacturing capacity are available. The use of antiviral drugs for prophylaxis or treatment (see previ- ous section of this chapter). The use of immunoprophylaxis for protection, including both non- specific approaches to stimulation of innate antiviral defenses and specific prophylaxis directed at particular pathogens. Nonspecific Inhibition of Virus Replication The first line of defense against viral infection is the innate immune response. Innate defenses not only act to control virus replication early after infection, but they also shape and influence the nature of subsequent spe- cific immune responses. This continuum between the innate and acquired immune responses to pathogens is increasingly being recognized. Many Or important components of the innate immune response contribute to the early control of viral replication. The best understood is interferon (IFN), a cytokine which is produced by many types of cells and was first recognized for its ability to make previously susceptible cells resistant to infection by a wide variety of viruses. In addition to the first recognized IFN, now known as type I or IFN odd, there are several other cytokines with important antiviral properties. Cytokines produced early after infection include type II IFN or IFN-^y, which is produced by natural killer (NK) cells, and tumor necrosis factor (TNF)y, which is produced by phagocytic cells such as mac- rophages. Current knowledge and therapeutic experience is most extensive for type I IFN, which induces an antiviral cellular state by interacting with the IFN alp receptor, IFNAR. IFNAR signals through a pathway involving transcription factors STAT-1 and STAT-2 to induce transcription of a large number of IFN-responsive genes and subsequent production of antiviral proteins. The best studied of these proteins and pathways are those involv- ing the dsRNA-activated protein kinase, PKR, which inhibits protein syn- thesis; the dsRNA-activated oligoaclenylate system, which degrades RNA; and the MX GTPases, which inhibit RNA synthesis. In addition to these direct antiviral responses, IFN also upregulates expression of major histo- compatibility complex (MHC) molecules on the cell surface, thereby en- hancing recognition from cells involved in inducing an immunologically specific immune response. From extensive study of these IFN-regulated pathways, several facts are clear: 1. The pathways described to date involve only a small proportion of the messages known to be induced by IFN (i.e., IFN ac/p probably induces about 90 different pathways).

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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 131 2. Each pathway affects replication to a different degree, depending on the particular virus. A pathway that interferes with the replication of one virus may have absolutely no effect on the replication of another virus. 3. Our understanding of how IFN inhibits viral replication is incom- plete. 4. Viruses have evolved a large number of mechanisms to counteract the effects of IFN. These mechanisms may or may not be preserved in the tissue culture-adapted virus strains most often used for study. Several recombinant forms of both IFN-a and IFN-p are currently available and licensed for treatment of a variety of diseases, including mul- tiple sclerosis, lymphoid tumors, and chronic viral infections (particularly hepatitis B and hepatitis C). Therefore, we have knowledge of dosing and side effects for prophylaxis against chronic infections in humans. However, our experience with prophylaxis against acute infections is very limited. IFN has been used locally for prophylaxis against upper respiratory infec- tions, and, although effective, open causes side effects resembling symp- toms of the disease being prevented. Many people would rather have a cold than suffer these side effects. Experience with preventing systemic infec- tions is limited to animal models, where efficacy can be demonstrated as long as the IFN or IFN-inducer (e.g., poly IC) is administered before or shortly after exposure to the virus. Therefore, although our experience with this approach is limited, prophylactic use of IFN is certainly a rational approach to protection from infection early after exposure. However, its effectiveness against the specific wild-type virus of interest would need to be confirmed. As mentioned above, viruses have evolved many ways to circumvent host cell antiviral activities (Alcami and Koszinowski, 2000~. For example, viruses from many different families (e.g., picornaviruses, rhabdoviruses, reoviruses, retroviruses, orthomyxoviruses, adenoviruses, herpesviruses, poxviruses) block the activation of the PKR pathway by either producing decoy RNAs, binding dsRNA, or degracling PKR protein. These mecha- nisms are especially prevalent in wi13-type viruses, whose ability to escape the effects of IFN is likely to be important for virulence and transmission. As another example, the virulent myxomatosis in the poxvirus family pro- duces proteins that bind host I-NF, IFN, and a broad range of chemokines. Because viral defenses against host innate immune responses are not neces- sary for viral replication in vitro, they may be lost, not expressed, or mu- tatect in tissue culture-adapted strains of virus. However, they are very important for in viva virulence. In addition to IFN alp, other less well-studied antiviral cytokines in- clude IFN-y and TNFa. In vitro, both exhibit antiviral activity against some viruses in some cells, but their effects are much more variable that those of

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132 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION IFN a/b. "Upstream" inducers of these effector cytokines, such as IL-12 or immunostimulatory oligonucleoticles, could potentially be developed as broadly active prophylactic agents. However, considerable research on tox- icity and effectiveness would need to be performed before any of these agents could be considered for widespread prophylactic use. Specific Inhibition of Virus Replication Acquired immune responses provide specific protection against re-in- fection by many viral pathogens and, as such, serve as the basis for protec- tion by immunization. In the pre-immunization era, immune globulin con- taining polyclonal antibodies to specific viruses was used for prevention and treatment for a number of infections (Ordman et al., 1944~. For both polio and measles, data from excellent controlled studies show that passive prophylaxis can prevent disease in outbreak situations; protection can last ~ ~ ~ 1 1 ~ r . 1 1 1 1 1 1 tor weeks alter a single nose. come or these data nave been used to deter- mine what levels of antiviral antibody need to be inducer! by vaccines in order to provide protection from infection. However, there are a couple of serious problems with passive transfer of immune globulin. First, the amount of antibody against the virus may be relatively low but the volume needed relatively large. This problem will be exacerbated as the population's immunity to the virus wanes following eradication and cessation of immunization. Second, using large pools of donors to generate the immune globulin carries the risk of transmitting other infectious agents. Fortunately, there has been considerable progress on this front since the early days when immune globulin was used for passive protection against polio and measles. This is illustrated by the current products available for prophylaxis against respiratory syncytial virus (RSV), a cause of serious lower respiratory disease in young infants, particularly those with cardiac and pulmonary abnormalities. There is no vaccine for RSV. Passive transfer of immune globulin is protective, but not all infants can tolerate the volume loads required to achieve protective antibody levels (PREVENT, 1997~. More effective and potent prophylactic products have been developed and licensed. In particu- lar, animal studies have shown that a mouse monoclonal antibody (MAb) provides protection against RSV by binding to the F protein. Determinants of antibody specificity lie in the variable complementary determining re- gions (CDRs) of MAb's Fab H and L chains. But the rest of the mouse MAb molecule induces an immune response in humans. Through genetic engi- neering, mouse MAb has been "humanized" so that every region of the molecule, except for those portions of the CDRs that determine specificity for binding to the RSV F protein, are now human. Humanized MAb pro-

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MEDICAL INTER VENTION AND TE CHNO ~ O GICAL SO ~ UTIONS 1 33 vices effective prophylaxis against severe RSV-inducec! disease (The Im- pact-RSV Study Group, 1998~. With recent technological improvements, variable regions of human antibodies with desired specificity can be cloned directly from antibody- secreting cells in the blood or bone marrow (Hoogenboom and Chames, 2000; Little et al., 2000~. These clones can then be cloned into another vector and converted directly into whole human IgG molecules Manna et al., 1999~. By knowing which antibody specificities are protective against eradicated viruses, clinically useful immunoprophylactic reagents could be generated relatively easily. These antibodies would then be available for production and use in the event of reintroduction or re-emergence of an eradicated virus. Conclusion Immunomodulators that would be broadly protective against viral in- fectionssuch as IFN alp, TNFa, and immunostimulatory DNA are in the early stages of development. IFN oc/p is the best characterized but has been used primarily for treatment of chronic viral infections, not prophy- laxis against acute viral infections. Humanized MAbs have proven success- ful at preventing acute viral infections and are currently being used as a prophylaxis against RSV. Technology has advanced to a point where spe- cific prophylactic MAbs could be developed for use against other viral pathogens besides RSV, but this has not been done for polio, measles, or smallpox. Given the diversity of viruses, it seems unlikely that a universal prophy- lactic agent will be identified. Rather, studies will need to focus on develop- ing prophylaxis for those infections deemed to pose the greatest risks. Prophylactic agents must be developed before they are needed in the post- . . erac Cation era. THE POTENTIAL ROLE OF PROBIOTICS AND MICROBIAL ECOLOGY IN HOST DEFENSE Susanna Cunningham-R2`ndles, Ph.D. Professor of Immunology, Department of Pediatrics Weill Medical College of Cornell University, New York, NY The human immune system provides host defense against sudden inva- sion from exogenous pathogenic microorganisms and viruses, while simul- taneously maintaining continual surveillance against incursion from endog- enous microbes. Immunization will create a specific pathogen-free

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134 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION environment only if there is a continuous normal immune response within an immunized majority of the community. An apparently healthy person's response to vaccine is often taken for granted, even though detailed knowI- edge of the normal immune response is lacking. There has been little con- sideration given to the possibility that the human immune system may be affected by selective pressure from changing world conditions. As immunity wanes, even immunized individuals may be highly vulnerable in the absence of total eradication. Immunity in the community at large is not determined by the poor response to some vaccines by young children and immuno- compromised persons if the proportionate representation of these groups is small. However, the increasing size of this poor response population for example in parts of the world with a high incidence of HIV infectionmay significantly affect whether standard immunization practices can lead to the eradication of infectious pathogens. The strength of the immune system is both challenged and maintained 3: , through continual interaction with an internal microbial milieu. Under- standing this fundamental interaction will provide new insights into what makes an immune response functional and will likely lead to novel ap- proaches to restoring or enhancing immune function. In healthy people, microflora are normally present on all external sur- faces and the internal surfaces of the upper respiratory tract, gastrointesti- nal tract, perineum, vagina and distal urethra. They are usually absent from the internal surfaces of the bronchi, alveolar spaces, urinary tract, and uterus, as well as the blood, deep tissues, organs, and brain. Within the gut, there are distinct, closely regulated differences in the relative density of bacteria. Mechanisms that mediate and maintain these regional differences include physical structures, such as the glottis; physi- ological barriers, such as gastric pH; and the continual action of both the innate and adaptive immune systems. The normal human gut is persistently colonized. Since there is no fixed boundary between colonization and infec- tion, response to persistent colonization likely involves repeated waves of immune activation. Thus, gastrointestinal colonization conditions the acti- vation potential of the immune system. The gut immune system operates independently of the systemic im- mune system, and the gut's resident T cells have developed specialized functional capacities independent of thymic influence. Recent studies (Gill et al., 2000; Macpherson et al., 2000; Walker, 2000) have shown that the gastrointestinal-immune interface is a frontier zone, and the gut's local innate response to antigenic or pathogenic challenge has a surprisingly strong influence on the systemic immune response. A key mediator for this response is the natural killer, or NK, cell. NK cells are characterized by their spontaneous ability to kill tumor or virally infected cells. They also produce cytokines, which regulate host defense against bacteria and influ-

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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 135 ence the development of the adaptive immune system. NK cells begin func- tioning at birth, when microbial colonization of the gut occurs. In the early post-birth period, neonatal NK cells show absent or decreased cytoly~cic activity against the reference erythroleukemia tumor cell target K562. How- ever, as demonstrated in Figure 5-1, certain bacteria can directly activate the neonatal NK cell system. This functional response is accompanied by he nova induction of gamma interferon procluction. The preparations of bac- teria used in these studies ImuVert (ribosomal vesicles from Serratia marscese?~s) and OK432 (whole inactivated Streptococcus pyogenes) have broad immunoadjuvant properties. These experiments in vitro mirror what happens in viva in response to conventional environmental microbes. This 30 25 Ox 20 a a ~ 15 a, 10 ~ O- . Endogenous ~ S. marscesens S. pyogenes ~ , ~ , rim ~ 1 50:1 25:1 Effecter: Target Ratio 12:1 FIGURE 5-1 Stimulation of neonatal natural killer cell activity by bacteria. This shows the effect of S. marscesens and S. pyogenes on NK activity of peripheral blood mononuclear cells in the short-term Cr5i release assay against K562. Data are given as percent specific release at three effecter target ratios. (Cunningham- Rundles and Nesin in "Persistent Bacterial Infections," 2000, reprinted with per- mission from ASM Press)

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136 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION unique response to bacteria probably evolved to provide a transition be- tween the pre-natal suppression of fetal immune effecter activity, which is necessary for the maintenance of maternal fetal tolerance, and the post- natal requirement for rapid response toward potential microbial pathogens. Microflora directly alter the architecture and physiology of the mucosa by inducing an immune response, which they probably continue to influ- ence and regulate throughout life. Emerging studies (Word and Adlerberth, 2000) have suggested that the specific composition of microflora is highly varied among different cultures and that it tenets to remain constant for the individual once established after birth. Normal flora do not directly harm the normal host; plus they contain commensals which produce nutrients, absorbable peptides, and vitamins, all of which benefit the host. It is now possible to stucly the potential significance of this lifelong interaction, thanks to the advent of genetic typing, which spurred investigation of flora com- prised of species resistant to current culturing methods. One study (Ahrne et al., 1998) showed that the we11-characterized beneficent commensals, such as lactobacilli, form a small and rather fragile part of the overall flora. The ecology of microflora is strongly influenced by oxygen tolerance. Commensal bacteria are primarily obligate anaerobes, whereas key patho- genic bacteria are facultative anaerobes that replicate faster in the presence of oxygen. Thus lactobacilli and bifididobacteria, which are normal gut commensals, survive and replicate in the presence of oxygen but not as effectively as, for example, E. coli. If beneficial microbes have a selective advantage, their colonization may prevent outgrowth of more pathogenic bacteria. Although Metchnikoff proposed in 1907 (Metchnikoff, 1907) that lactic acid bacteria would have a favorable effect on health, the concept of probiotic bacteria living mi- crobes introduced into the body to improve intestinal microbial balance is recent (Fuller, 1989~. Probiotic bacteria have proven effective against anti- biotic-associated diarrhea and certain persistent and clinically significant infections, such as C. difficile. Experimental studies (Bergogne-Berezin, 2000; Hirayama and Rafter, 1999; Hove et al., 1999; Kirjavainen et al., 1999; Majamaa et al., 1995) have shown that probiotic lactobacilli can neutralize carcinogens, replace microflora that produce carcinogens and tumor promoters, and produce antitumor factors through direct actions in the gastrointestinal tract. Essential characteristics for efficacy include resis- tance to acid and bile and ability to colonize and adhere to the colonic mucosa (Bengmark, 1999~. Moreover, current studies (Cunningham- Rundles et al., 2000; Devi et al., 1999; Hessle et al., 1999) have suggested that probiotic lactobacilli may serve as immunoadjuvants, thereby increas- ing weak systemic immune response, even in the HIV+ host. Possible mecha- nisms of action include competition for specific ecological niches, immuno- ~:

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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 13 7 logical stimulation of the mucosal barrier, and induction of specific cytokine patterns. Thus, probiotic bacteria have strong potential to sustain natural im- mune response towards environmental pathogens in the post-vaccine era. Additionally, probiotic lactobacillus may prove useful in strengthening im- mune responses in persons whose host defense capacity has been compro- mised by chronic infection or short-term stressors. However, there are a few key questions concerning the use of probiotic bacteria in the immuno- deficient host, including: . Can the immune-deficient host develop a normal immune response towards lactobacitIus? Is this response qualitatively or quantitatively different from that of immunocompetent persons? . Are there safety issues, such as potential for transiocation? The most extreme example of acquired immune deficiency is HIV infec- tion. Normal bacterial flora are altered in HIV infection, as evident by the frequency of bacteremia associated with altered gastrointestinal function, diarrhea, and malabsorption. Failure-to-thrive is relatively common in con- genital HIV infection and is linked to altered gastrointestinal function and chronic cytokine activation. Our lab studies the effect of L. piantarum 299v, a specially developed probiotic lactobacillus, on growth and specific systemic immune response following oral supplementation in the HIV+ child. There appears to be a generally beneficial effect on immune response. Surprisingly, the HIV+ children's level of cross-reacting immune response to LP299, as a group prior to supplementation, is essentially independent of CD4+ T cell percentage, which is unlike response to any other activator. Data are shown in Figure 5-2. Children who did not respond to LP299v before supplementation did develop response after supplementation; the oral supplement was well tolerated, colonization was temporary, and there were no side effects. The mechanism of action is currently under study; preliminary data suggest that treatment promotes a T helper type 1 cytokine response. These studies support current interest in commensal bacteria as anti- gen-delivery vehicles, as well as potential adjuvants. The possibility that modulation of gastrointestinal flora might be used to strengthen immune response is especially relevant for protection of future populations against emerging infections in a post-immunization era where, paradoxically, the immune system may face even greater challenges.

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138 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION 3500 3000 - 2500 - 2000 - 1 500 1 000 500 - O - CD4+ T cell = 7% CD4+ T cell = 34% . HIV+ children FIGURE 5-2 Immune response to Lactobacillus plantarum 299v in HIV+ children in relationship to CD4+ T cells. Peripheral blood mononuclear cells were cultured in a microtiter plate assay and pulse labeled with 3H thymidine. Data show mean net maximum response to LP299v antigen in children grouped by CD4+ T cell level. (Cunningham-Rundles and Nesin in "Persistent Bacterial Infections," 2000, reprinted with permission from ASM Press.) REP ERENCES Ahrne S. Nobaek S. Jeppsson B. Adlerberth I, Wold AK, and Molin G. 1998. The normal Lactobacillus flora of healthy human oral and rectal mucosa. Journal of Applied Micro- biology 85:88-94. Alcami A and Koszinowski UH. 2000. Viral mechanisms of immune evasion. Trends in Microbiology 8:410~1 8. Bengmark S. 1999. Gut microenvironment and immune function. Current Opinion in Clinical Nutrition and Metabolic Care 2:83-85. Bergogne-Berezin E. 2000. Treatment and prevention of antibiotic associated diarrhea. Inter- national Journal of Antimicrobial Agents 16(4):521-526. Centers for Disease Control and Prevention. 2000. Outbreak of poliomyelitis- Dominican Republic and Haiti, 2000. Morbidity and Mortality Weekly Report 49:1094, 1103.

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140 CONSIDERATIONS FOR VIRAL DISEASE ERADICATION Shulman EM, Manor Y. Handsher R. Delpeyroux F. McDonough MJ, Halmut T. Silberstein I, Alfandari J. Quay J. Fisher T. Robinov J. Kew OM, Crainic R. and Mendelson E. 2000. Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel. Journal of Clinical Microbiol- ogy 38(10):3729-3734. Walker WA. 2000. Role of nutrients and bacterial colonization in the development of intesti- nal host defense. Journal of Pediatric Gastroenterology and Nutrition 30:S2-S7. Wold A and Adlerberth I. 2000. Pathological consequences of commensalism. In Nataro JP, Blaser M], and Cunningham-Rundles S. (eds.). Persistent Bacterial Infections, pp. 145- 163. Washington, DC: American Society of Microbiology Press. World Health Assembly. 1988. Global Eradication of Poliomyelitis by tile Year 2000. Geneva: World Health Organization. Yoshida H. Horie H. Matsuura K, and Miyamura T. 2000. Characterisation of vaccine- derived polioviruses isolated from sewage and river water in Japan. Lancet 356(9240):1461-1463.