AIDS

Solving the Molecular Puzzle

by Michelle Hoffman

In his 1993 inaugural address, President Clinton warned about the twin enemies of "ancient hatreds and new plagues" that continue to threaten and shape the world. There can be no doubt that among those plagues the new president was thinking of AIDS.

ANCIENT HATREDS AND NEW PLAGUES

Acquired immunodeficiency syndrome, or AIDS, is very much a modern plague, a reflection of the times in which it emerged. In a world where cultural and economic boundaries between countries are less firm than geographic ones, the grim reality is that epidemics respect no borders. AIDS is a plague that could emerge only in a modern world where, for better or worse, rapid mass transportation links materials, people, and pathogens from all over. And so it is that the human immunodeficiency virus (HIV), the agent that causes AIDS, could first evolve in a remote corner of the world and within little more than a few decades become a dread fear in every corner



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A Positron Named Priscilla: Scientific Discovery at the Frontier AIDS Solving the Molecular Puzzle by Michelle Hoffman In his 1993 inaugural address, President Clinton warned about the twin enemies of "ancient hatreds and new plagues" that continue to threaten and shape the world. There can be no doubt that among those plagues the new president was thinking of AIDS. ANCIENT HATREDS AND NEW PLAGUES Acquired immunodeficiency syndrome, or AIDS, is very much a modern plague, a reflection of the times in which it emerged. In a world where cultural and economic boundaries between countries are less firm than geographic ones, the grim reality is that epidemics respect no borders. AIDS is a plague that could emerge only in a modern world where, for better or worse, rapid mass transportation links materials, people, and pathogens from all over. And so it is that the human immunodeficiency virus (HIV), the agent that causes AIDS, could first evolve in a remote corner of the world and within little more than a few decades become a dread fear in every corner

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A Positron Named Priscilla: Scientific Discovery at the Frontier of the world. Today, more than 600,000 people worldwide have been diagnosed with AIDS (see Figure 3.1), and in the United States alone at least 1 million people are infected with HIV (see Figure 3.2). Throughout the world, still more are infected and have yet to know or have yet to develop the symptoms of AIDS. But numbers alone do not reflect the dimensions of the "world AIDS crisis," as President Clinton called it. Certainly, more people have had flu from influenza virus than have suffered from AIDS. What distinguishes AIDS from every infectious disease to come before it is the scientific challenge it represents to a research community that has successfully controlled all of the plagues of the past. Hardly anyone living in the United States today can remember a time when they were concerned about typhoid or tetanus. Few children today will grow up with the threat of polio or smallpox. Improved public health measures and potent vaccines have made these diseases the exception rather than the rule in the United States and in most of the industrialized world. In more recent memory, both toxic shock syndrome and Legionnaire's disease emerged and were vanquished by science within a few short years. Against this backdrop of success fighting infectious diseases, comes HIV, a virus whose natural history is so unusual that no prior experience with any infectious agent has appropriately prepared modern science to tackle it. And yet no previous era has been better prepared to take on the challenge. Only now does science have the experimental tools to unravel the details of the peculiar life style of this virus. It is one of the greatest ironies of this epidemic that stopping it will owe as much to the technology of the era as its spread. The hallmark of AIDS is the slow but complete erosion of the immune system. Years, sometimes many years, after a person becomes infected with the virus, his or her immune system is so weakened that it is unable to fight off the bacterial and viral assaults that a strong immune system could easily defeat. For that reason people with AIDS are vulnerable to a host of diseases rarely found in the general population. These are called opportunistic infections because they take the opportunity to strike someone with depleted immune defenses. Ultimately, the patient dies from the effects of one of these diseases. The list of opportunistic infections that most frequently affect AIDS patients itself reads like a catalog of plagues. Tuberculosis, recurrent

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A Positron Named Priscilla: Scientific Discovery at the Frontier FIGURE 3.1 Cumulative number of reported adult AIDS cases worldwide through late 1992. Map derived from data provided by the World Health Organization, 1993. pneumonia (caused by a parasite called Pneumocystis carinii), and a rare skin cancer called Kaposi's sarcoma are some of the most common causes of death for people with AIDS in the United States. Other AIDS-related conditions include lymphoma and other cancers, systemic yeast infections, toxoplasmosis and other degenerative disorders of the nervous system, and an unexplainable wasting syndrome (see Table 3.1). If any one of these conditions in itself is not fatal, the cumulative effect of fighting off so many infections with an increasingly damaged immune system often is. In one sense the solution to the problem is clear. If science could find a way to lessen the immune-depleting effects of the virus, people with AIDS would not be so defenseless against opportunistic infections. But this strategy is not as simple as it sounds. Once the virus infects an individual, the virus and immune system are locked in an intimate and paradoxical relationship. Following infection the virus becomes part of the immune system and in so doing undermines the very defenses that are supposed to fight it. Eliminating the virus therefore means turning the immune system against itself. And there lies another of the disease's ironies and scientific challenges.

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A Positron Named Priscilla: Scientific Discovery at the Frontier FIGURE 3.2 Estimate of HIV infections in adults as of late 1992. Source: World Health Organization, 1993. The ultimate goal of current AIDS research is to extricate the virus from the immune system without causing further damage. To do that scientists over the past decade have focused on the complex relationship between the virus and the cells of the immune system as well as relationships of the different immune cells to each other. VIRUSES A famous scientist once defined a virus as "a bit of bad news wrapped in protein." Indeed, while the experimental study of viruses can unlock a wealth of scientific information, viruses offer little else of value to human beings. Unlike the occasional bacterial species that can provide a beneficial service to humans (e.g., bacteria found lining the human intestines that aid digestion), viruses are never Good Samaritans. They seem perfectly willing to take up residence in the human body without doing anything to earn their keep. But the scientist's comment also touched on the very simple structure of a virus. Compared with the complexity of the mammalian or bacterial cell, viruses are remarkably minimal. Most viruses are

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A Positron Named Priscilla: Scientific Discovery at the Frontier TABLE 3.1 Conditions Included in the 1993 AIDS Surveillance Case Definition • Candidiasis of bronchi, trachea, or lungs • Candidiasis, esophageal • Cervical cancer, invasive* • Coccidioidomycosis, disseminated or extrapulmonary • Cryptococcosis, extrapulmonary • Cryptosporidiosis, chronic intestinal (> 1 month's duration) • Cytomegalovirus disease (other than liver, spleen, or nodes) • Cytomegalovirus retinitis (with loss of vision) • HIV encephalopathy • Herpes simplex: chronic ulcer(s) (> 1 month's duration); or bronchitis, pneumonitis, or esophagitis • Histoplasmosis, disseminated or extrapulmonary • Isosporiasis, chronic intestinal (> 1 month's duration) • Kaposi's sarcoma • Lymphoma, Burkitt's (or equivalent term) • Lymphoma, immunoblastic (or equivalent term) • Lymphoma, primary in brain • Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary • Mycobacterium tuberculosis, any site (pulmonary* or extrapulmonary) • Mycobacterium, other species or unidentified species, disseminated or extrapulmonary • Pneumocystis carinii pneumonia • Pneumonia, recurrent* • Progressive multifocal leukoencephalopathy • Salmonella septicemia, recurrent • Toxoplasmosis of brain • Wasting syndrome due to HIV * Added in the 1993 expansion of the AIDS surveillance case definition. SOURCE: Centers for Disease Control, January, 1993 nothing more than a shell of protein that contains a packet of genes on the inside. Viewed externally, each HIV particle is shaped like a 20-sided soccer-ball (see Figure 3.3). The virus takes its form from the protein making up its outer shell, which is sometimes called the capsid. Overlying the outer capsid is a lipid membrane, pilfered from the human host cells that the virus infects. Poking through the capsid and the membrane are proteins studded with sugars, called glycoproteins. The glycoproteins on HIV are often depicted as lollipop shaped. The stick of the lollipop is made of a glycoprotein with a molecular weight of 41 kilodaltons, and, according to the convention of referring to proteins and glycoproteins by their molecular weights, it is named gp41. The "candy" of the lollipop is a

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A Positron Named Priscilla: Scientific Discovery at the Frontier glycoprotein of 120 kilodaltons, called gp120. Because gp41 and gp120 are initially associated in a single protein, scientists refer to this precursor form as gp160. Contained within the outer shell is another protein capsule. This cone-shaped capsule, called the core, is made up of many protein molecules, each with a molecular weight of 24 kilodaltons, named, appropriately, p24. Each component of the structure serves a distinct purpose. The glycoproteins gp41 and gp120 are known to be crucial in anchoring the virus to the cell it will infect, while the lipid membrane helps the virus enter that cell. The capsid protects the virus and gives it shape. And all of this protective packaging is designed to safeguard the precious cargo found within the viral core. There the virus places its hope for the future: Its genes and a few proteins that will help it carry out its reproductive mission. Because viral structure is so pared down, many scientists have argued that viruses cannot even be considered living organisms. To be alive in the biological sense, an organism must have the capacity to feed itself and replicate itself. Viruses can do neither on their own. The viral genes provide the instructions for making more viruses. They are the guidelines for the manufacture of structural proteins that make up the FIGURE 3.3 The structure of the Human Immunodeficiency Virus (HIV). At the center of the virus is a cone-shaped core that contains the viral genetic material and auxiliary proteins required for viral replication. The cone sits within a protein shell, called the capsid, and the entire assembly is surrounded by a membrane, pilfered from the human cell in which that particular virus particle was replicated. Sticking through the membrane are the viral proteins that allow the virus to anchor onto and then infect a new cell. (Adapted from Gelderbloom et al., 1987. Virology, Volume 156, pp. 171–176.)

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A Positron Named Priscilla: Scientific Discovery at the Frontier virus's capsid and core and enzymatic proteins that reproduce the viral genes and help package them into the shell. But the actual work of manufacturing the genes and proteins needed to produce more viral particles is achieved within the infected host cell, using host cellular machinery, energy, and the raw materials—the chemical subunits—found within the cell. The production of new viral particles often means that the host cell must turn away from its ordinary business and devote itself entirely to the assembly of new viral particles. Viral dependence on the host cell requires that at some stage in the virus's life cycle it must enter the host cell and there fulfill its biological mission to reproduce. And so it is that newly manufactured viral particles released into the blood must find new host cells to infect, so that they too can start to reproduce. Viruses must spread from cell to cell within the infected individual and to new individuals, in search of new cellular hosts that provide the facilities for them to meet their reproductive needs. THE IMMUNE SYSTEM Healthy people are anything but defenseless against microbial invaders. On the contrary, the human body can be viewed as a veritable fortress. The skin provides a strong and almost impenetrable barrier to infection, and saliva is full of degradative enzymes. If, however, a microbe should get past either of these barriers, it could well end up in the lungs, where it might induce the infected person to cough. Coughing tosses the microbe up the windpipe and down the esophagus where it will land in the stomach. From the point of view of a microbe unfortunate enough to end up there, the stomach is nothing more than a vat of harsh acids and more degradative enzymes. Needless to say, this points to sure destruction for the microbe. As effective as these barriers usually are, the human body is not completely impregnable to pathogens, and people do get sick. In the event a pathogen does get past the body's defensive barriers, humans also have at their disposal an internal system for removing invasive pathogens. It is this internal system—composed of cells and the chemicals they secrete—that is referred to as the immune system. The immune system is often compared to the military, and rightly so. Like soldiers, the cells of the immune system are designed to recognize foreign invaders and attack them. But the analogy extends even farther. In both systems, protective forces are deployed to the site of attack to fight in the same mode as the attackers. For example, military

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A Positron Named Priscilla: Scientific Discovery at the Frontier forces are prepared to respond to attacks in the air, sea, or on land, depending on where the enemy threatens. Pathogens, like enemy armies, have different modes of attack, and the immune system, like the military, has specialized divisions to fight back in the relevant mode. Broadly speaking, pathogens invade the body in two modes. Some pathogens live in the body's fluids but remain outside the host cells. These are called extracellular pathogens. Other pathogens are intracellular. Intracellular pathogens spend at least part of their life cycles inside host cells. As a general rule, these two modes of attack correspond to the two major classes of infectious disease-causing agents: bacteria and viruses. Most bacteria are extracellular pathogens. As such, they live and thrive outside the host cells. For the bacteria the host is a sort of incubator and a source of nutrients, providing a warm and fertile environment for the bacteria to grow and multiply. But most bacteria are self-sufficient in at least one regard. They contain within themselves all of the cellular machinery required to multiply. Reproductively independent as they are, bacteria can in some but not all cases be less than benevolent to their human hosts. During the course of their life cycles, bacteria can manufacture and secrete biochemicals that are toxic to their hosts. Even though they remain outside host cells, bacteria can still cause disease via their toxins. In contrast to the relatively independent bacteria, viruses spend only part of the time outside the host cells. When it comes time to multiply, they must enter the host cells. While viruses contain a complete set of their own genes, they carry little else inside their protein shells. Without any means to replicate their own genes or manufacture the proteins that encase those genes, viruses are compelled to enter a person's cells, making the infected person's cells an unwilling host to an all too often pernicious viral guest. The unwanted viral visitor co-opts host cellular machinery to carry out its own gene and protein synthesis. Viruses are therefore intracellular pathogens. Since pathogens can exist either intracellularly or extracellularly, the immune system must be ready to respond in either context. Or, returning to the military analogy, the immune system must have at least two branches: one that is prepared to fight extracellular pathogens and another to fight intracellular ones—and indeed it does. To fight extracellular pathogens, the body produces antibodies. These highly specific molecules are manufactured and secreted into the blood by a type of white blood cell called a B-cell. Once in the blood the antibodies can fight infectious agents that are swimming there. The

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A Positron Named Priscilla: Scientific Discovery at the Frontier blood can also help circulate the antibodies to other tissues in which bacteria and newly released viral particles might be found. Antibodies are extremely specific in what they recognize. A healthy person manufactures antibodies that recognize substances foreign to the body but not the body's own chemicals. Each antibody picks out only a small portion of a single molecule on the surface of a bacteria or a virus. But that is sufficient for the antibody to bind to that molecule, and eventually many antibody molecules coat the pathogen's surface. The antibody-coated pathogen then becomes a target for destruction by macrophages, another cell type in the immune system. Fighting intracellular pathogens is a trickier business. Because the pathogens gain entry to and are hidden within the body's own cells, infected cells look to the immune system just like every other host cell. But infected cells are not without their defenses. All cells have a kind of housekeeping system that digests particular proteins when they are no longer needed. In some instances, protein fragments, called peptides, made during this digestion process are carried out to the cell's surface and held there by surface proteins. When peptides derived from the host's own proteins are displayed on the cell surface, they are ignored by the immune system, which has a mechanism for distinguishing proteins of the host from foreign proteins. In an infected cell, peptides from the pathogen are also transported to and displayed on the cell's surface. These foreign peptides serve as a distress signal that tells the immune system that the host cell in question is harboring a pathogen. The immune system responds to the distress call by deploying another type of white blood cell, a T-cell, which is specially equipped to detect the foreign peptides on the surface of the infected cells. Once the infected host cell has been identified, the T-cell destroys the host cell along with its infectious cargo. Because of its activity, this type of T-cell is called a cytotoxic T-lymphocyte (CTL) or, more colloquially, a ''killer" T-cell. It is interesting to note that under a microscope the antibody-secreting B-cells and killer T-cells are almost indistinguishable. B-cells are distinguished from T-cells first by their site of maturation. B-cells mature in the bone marrow, T-cells in the thymus. But beyond that, B-cells and T-cells carry different proteins on their surfaces and manufacture and secrete different sets of biochemicals that help them carry out their different functions. Like any well-organized military system, the immune system is tightly coordinated. In this defense network the generals are another type of T-cell, called a "helper" T-cell (see Figure 3.4). By secreting the

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A Positron Named Priscilla: Scientific Discovery at the Frontier FIGURE 3.4 The immune system relies on the concerted activities of a number of different cell types. At the center of this activity is the helper T-cell, which receives information about invading pathogens from macrophages and stimulates pathogen-fighting activities in other immune cell types. The helper T-cell will trigger antibody production in B-cells and will activate "killer" T-cells, which destroy other cells in the body that are infected with the pathogen. Unfortunately, the helper T-cell is the target for infection with and destruction by HIV. As the number of helper T-cells decreases throughout the progression of AIDS, the person's immune system becomes depleted and unable to fight off pathogens effectively. proper chemical signals at the appropriate time, helper T-cells can boost the populations of B-cells that secrete a particular antibody or they can increase the number of killer T-cells that recognize and eliminate virus-infected host cells. As such, helper T-cells are pivotal in plotting, coordinating, and implementing the defense strategy against both intra- and extracellular pathogens. Viruses and bacteria have evolved many complex mechanisms to dodge immune detection and destruction. Pathogens have been known, for example, to use camouflage, cloaking themselves in proteins that resemble the host's own. Other pathogens use changeable disguises. As soon as the immune system learns what the invader looks like, the pathogen has changed its chemical "face," which the immune system has

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A Positron Named Priscilla: Scientific Discovery at the Frontier to learn to recognize anew and which leaves it continually trying to play "catch up" with the pathogen. To be sure, HIV employs some of these guerilla tactics in its strategy to avoid the immune system, too. But this virus goes beyond that. HIV, it seems, does not merely dodge the immune system, it destroys it. An invader wishing to destroy host immune defenses can choose one of two ways to do it. The invader can kill each of the soldiers in the military or it can take hold of the command center. An immune system may have a complete set of soldiers—in this case B-cells and killer T-cells—but still remain immobile if the appropriate command never goes out to activate it. And the proper command might be impaired if something were to happen to the helper T-cells. As it turns out, HIV has a special affinity for and may be particularly harmful to the helper T-cells. While many of the interactions between HIV and the immune system are being worked out, and scientists are still debating how each of these interactions contributes to AIDS, most researchers will agree that part of the answer lies in the special relationship between HIV and the helper T-cell. LIFE CYCLE OF HIV Initial Contact, the Receptor The association between viruses and host cells begins when the virus tries to enter the cell. Because this is a crucial step in viral replication and it starts an infected individual on the road to developing a disease, a great deal of scientific investigation has focused on this process. Obviously, no host cell willingly invites a virus inside it. And yet viral access to host cells by no means constitutes a forced entry. Viruses use a kind of chemical subterfuge to gain access to their hosts. In the most common scenario, a virus poses as something that would normally be admitted to the cell and uses the normal ports of entry. This method is not unlike a thief who pretends to be a locksmith or an appliance-repair person to enter a house targeted for robbery. The cellular equivalent of a door is a surface protein called a receptor. A host cell has on its surface a great number of receptors that help the cell sense and interact with its external environment. To be sure, not all receptors are doorways. Most receptors receive external chemical messages and then initiate some sort of internal cellular response. Locomotion, gene expression, or protein synthesis and secretion are often touched off when a receptor on the cell's surface receives the

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A Positron Named Priscilla: Scientific Discovery at the Frontier possible that Tat secretion initiates a spate of viral reproduction in previously latently infected cells. In addition, there are some indications that Tat may directly kill uninfected cells. Alternatively, Tat may also alter the chemical signals that are sent out from a helper T-cell. According to this theory, the altered signals result in an immune response that is ineffective or inappropriate. For example, there may be too many signals stimulating antibody production and not enough stimulating killer T-cell activity, so that while there is an immune response it is ineffectual and does not eliminate the virus. New evidence has come to light to suggest that the Net protein may also be secreted. Some scientists think that Nef may act as a so-called superantigen. Superantigens are thought not to depress immune activity but to overstimulate it. As a consequence, the immune system starts to look like a ''Keystone Cops" episode where the perpetrator slips past a frenzied and disorganized immune system. It is important to note that many of these phenomena are studied in the laboratory using viruses and immune cells that have been cultured outside the human hosts from which they were taken. The artificial circumstances of culture conditions may well alter some of the biochemistry of these entities, and scientists caution that the behavior of cells and viruses may be different in intact living systems. So the question of whether the laboratory situation realistically reproduces real life must be addressed by scientists hoping to understand the relationship between HIV and AIDS. But even if all of these phenomena occur in a person with AIDS, scientists must also assess which mechanisms are of primary importance in helper T-cell death and which are fairly minor contributors. New evidence comes to light almost daily that makes the research community evaluate these questions in a new way. Some scientists believe that as the virus replicates within a host new strains emerge that are more efficient killers, more aggressive at reproducing, and more effective at infecting cells than its progenitors. Those who believe this suggest that the emergence of these potent new strains accounts for the sudden and rapid decline of the immune system after 10 or more years of calm. As research techniques improve, scientists discover aspects of the interaction they could not previously discern. For example, using higher-resolution techniques, some research groups are finding that many more than 1 percent of helper T-cells are actually infected with HIV. New estimates suggest that 20 to 30 percent may be infected at any one time. Some researchers think these numbers are adequate to explain the observed loss of helper cells. In that case the indirect mechanisms of

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A Positron Named Priscilla: Scientific Discovery at the Frontier killing may add to immune destruction but may play a relatively minor role. Although not all scientists agree on the relative contributions of these various mechanisms to the development of AIDS, they are in agreement that there may yet be facets of HIV that remain to be uncovered. Some hope that a definitive answer might lie in these still undiscovered interactions. VACCINES AND THERAPEUTICS Scientists are interested in the natural history of the virus in part because it is so unusual and its study promises to unlock many mysteries about the different ways that evolution has invented to do things. Take, for example, the unexpected finding of reverse transcription, which taught scientists that information could flow in more than one direction. Beyond satisfying intellectual curiosity, the intense interest that HIV and AIDS have received is motivated by a deep desire by the scientific community to eradicate HIV and AIDS. In the past, science has eliminated plagues with vaccines and drugs, and it is looking toward these to deal with HIV and AIDS. While people can try to avoid contact with infectious agents, some infection is inevitable. Most encounters with pathogens are completely random, and while no one can control when they might run across a virus or a bacterium that will cause them to get sick, vaccines can help them prepare in advance to fight the infection before it gains access to their system. The immune system is a defense network. Like any defense system, it needs to know what its enemy looks like, so it knows what to attack. Of course, after infection, once an invader has made its way into a person's system, the immune system will learn to recognize it and will respond by producing antibodies and deploying killer T-cells. But mobilizing such an attack takes time. The immune system needs to decipher the features of the foreigner and discern that the pathogen is in fact foreign. While the immune system is doing that, the pathogen is multiplying and causing disease. With vaccines, physicians try to eliminate or at least reduce the lag time so that the immune system is ready to kill an invading pathogen at the moment it enters the body, before it can infect cells, multiply, or cause disease. The vaccine is a device to teach the immune system of an uninfected person the features of a pathogen before that person ever comes in contact with it. The concept is identical with hanging a "Wanted" poster in public places, so criminals will be recognized and apprehended before they commit another crime.

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A Positron Named Priscilla: Scientific Discovery at the Frontier Vaccines In immunological terms there are several ways of hanging a "Wanted" poster. But the basic idea in all vaccine strategies is to inject some portion of the pathogen into the blood where immune cells will encounter it and learn to recognize it. Some small portion of these immune cells will always remain in the blood whether or not the pathogen is ever encountered. These few cells constitute what is called immunological memory, because they remember what the pathogen looks like. If a vaccinated person is challenged by the real pathogen, these few cells will proliferate, producing many more cells like them that can either kill infected host cells or secrete antibodies against the pathogen. In effect, the vaccine allows a person to respond to a pathogen much more quickly than he or she could if their immune system had no prior experience with the pathogen. The trick in developing a vaccine is to choose the correct portion of the pathogen, one that will elicit an appropriate immune response. For example, if one were to choose a protein fragment from the pathogen that was identical with a protein fragment found in the host, the immune system would not identify the fragment as foreign and would not bother to mount an immune response against it. Since it is difficult to know in advance what the immune system will see as foreign, the traditional approach to antiviral vaccines has been to expose the immune system to the entire virus. Naturally, viruses used in vaccines must be modified so that they do not cause disease. One form of polio vaccine, for example, makes use of viral particles that have been killed. Injecting these killed polio virus particles helps a person's immune system to become familiar with the features of the polio virus and develop immunological memory to it. But the killed vaccine itself cannot infect cells, reproduce, or cause disease. As an alternative to using killed virus, some vaccines are made of live virus. Even if the virus is live, however, it has been modified in such a way that its potency is attenuated. It cannot multiply or cause disease. A second form of the polio vaccine uses live attenuated virus. Both approaches are being explored experimentally against HIV, using animal models. While some research groups have reported limited success, it must be stressed that they have employed very special conditions, and critics of these experiments point out that the experimental conditions are a far cry from those presented in the real world. Many scientists are also concerned about the advisability of using either approach, but especially live attenuated virus, when dealing with a virus as harmful as HIV. The attenuated virus is genetically disabled so that

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A Positron Named Priscilla: Scientific Discovery at the Frontier theoretically it cannot reproduce, but skeptics question whether it might spontaneously mutate back to a form that can reproduce, which would make a vaccinated person sick rather than immunologically prepared to fight disease. The slight chance of such a mutation makes most people very cautious when proposing the use of live attenuated vaccines against HIV. Newer approaches to vaccine development aim at skirting the safety issue. Because some researchers are uncomfortable with the idea of using either dead or attenuated whole virus, creative new vaccines, which are as yet untried in humans, offer protein fragments or peptides derived from the virus. Since there is no chance that new viral particles can be generated from peptides, they are deemed safer, at least in that respect, than the classical whole-virus vaccines. Candidate peptides include segments from the surface glycoproteins, but scientists are experimenting with a number of peptides derived from other viral proteins, like the reverse transcriptase and the core proteins. Even though these proteins are found on the inside of the virus, they are chewed up by the host cell following infection, and some of the resulting peptides are displayed on the infected cell surface, detectable by the immune system. As such, it may be worthwhile to train the immune system to recognize them as well as proteins on the viral exterior. In the case of HIV, however, safety is only one of the concerns of vaccine developers. The concept of a vaccine assumes that the virus used to make it looks very much like the real virus that a vaccinated person might encounter. But scientists have learned not to make that assumption with HIV because it is highly mutable. Some portions of the gp120 molecule, for example, can change over just one reproductive cycle, so in the course of a year a virus may not at all resemble the original infecting virus, at least not in one of these highly changeable segments of gp120. Viral mutability poses a frustrating roadblock to vaccine development. It seems that the variable regions of gp120 are highly effective in activating an immune response, so scientists want to make use of them in vaccines. But because they change so rapidly, the immune system is always lagging behind the viral disguise, looking for a face that has already changed. Sadly, the regions of gp120 that cannot change because they are required for the interaction with the CD4 receptor are not as good at stimulating the immune response. A truly effective viral vaccine would therefore have to anticipate all of the viral variants a person is likely to encounter and include a mixture of them so that the immune system can get to know all of them. That, scientists say, is simply not yet possible.

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A Positron Named Priscilla: Scientific Discovery at the Frontier Even if both the safety and the viral variability issues could be overcome, HIV presents one final challenge to vaccine developers. The immune system can fight only enemies that it can see, but HIV creates many unseen enemies. The virus has a latency period, where the host cell is infected, harbors viral genes and has the potential to reproduce new viral particles at any moment but does not. In this quiescent state the infected cell displays no viral peptides on its surface and has no way of signaling its distress. Developing a vaccine that will distinguish infected but quiescent cells from uninfected cells is an intellectually imposing task. In one particularly innovative approach, peptides are administered consistently over time, so that the immune system remains at a high state of readiness against HIV. The hope here is that a highly alert immune system might be able to kill off the virus swiftly, either to avert initial infection or to quell reproduction of new viral particles that emerge as latent cells become activated. The strategy taken in this approach is to package the genes for the immunologically stimulatory peptides inside another organism that will continuously produce them and "remind" the immune system what the HIV perpetrator looks like. The way this vaccine works is that genes for some HIV proteins are placed inside a single-celled organism called BCG, a harmless derivative of the bacillus that causes tuberculosis. As a vaccine against tuberculosis, BCG has already been widely used and proven safe in humans. What makes it especially useful for vaccines where it is desirable to maintain a high level of immunological readiness is that the organism is not killed. Rather, the immune system isolates the bacillus in a ball of cells called a granuloma. Even though BCG is held prisoner inside the granuloma, it can still produce substances and secrete them into the blood. Potentially, BCG can be engineered to secrete HIV peptides for an individual's entire life. Thus, if the strategy works, it carries the potential for imparting lifetime immunity. Like all of the other vaccine strategies, however, this one is still in the early experimental stages and is not yet ready for human use. Therapeutics As creative as vaccine research has been, scientists who work on AIDS are generally more optimistic about new advances in treating people who already have AIDS with drugs than in preventing infection with vaccines, at least for the immediate future. Broadly speaking, drugs aimed at combating HIV can do one of two things: they can augment the

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A Positron Named Priscilla: Scientific Discovery at the Frontier immune response against the virus to try to kill off as many viral particles and virally infected cells as possible, or they can sabotage the reproductive efforts of the virus so that no new particles are made. Drugs that boost the immune system are similar to vaccines, and in many cases employ the same strategies, only they are administered after the person has become infected. In fact, some vaccines are being tested on people with AIDS to see whether they prolong the asymptomatic period or otherwise halt progression of the disease by boosting the immune response. In theory, all of the vaccines under development are potentially useful in treating people after they become infected or after they develop AIDS. In addition to the vaccines already discussed, which all seek to elicit antibody and killer T-cell production, scientists are also testing the possibility of giving antibodies directly to patients who are HIV seropositive and to those who already have AIDS. The problem with the antibody approach, say scientists, is viral mutability. If an antibody is given that helps the immune system kill off one viral variant, many more slip by undetected. Most scientists are coming to the conclusion that the most effective antiviral strategies combine many different activities. In other words, therapeutic approaches of the future may well combine drugs that boost the immune system with ones that inhibit viral replication. A New Generation of Drug Treatments The general hope is that a new generation of drugs may help extend the asymptomatic period from 10 to 30 years or more. While this is not as desirable as completely eliminating the virus from a person's system, it will allow an HIV seropositive individual to enjoy a longer disease-free life and may also limit the severity of any symptoms should they develop. Antimicrobial drugs each work differently, but they all share the same fundamental goal—to sabotage the biochemical machinery that produces the progeny of the infecting microogranism. Since each machine is different, each therapeutic monkey wrench must be tailor-made to the machine it is trying to inhibit. A big problem in drug development, however, is that the host cellular machinery is often very similar to the machinery used by the replicating virus or bacteria. In fact, viruses are heavily dependent on the host cell's machinery to carry out their reproduction. So drug developers walk a tightrope trying to find agents that harm specifically microbial machines but that leave cellular machines intact. To the degree that drugs fail to be specific for their microbial targets, they cause side effects. Sometimes a drug that is very

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A Positron Named Priscilla: Scientific Discovery at the Frontier potent against a pathogen is also severely toxic to the host and is therefore not usable. With the hope of finding a balance between efficacy and toxicity, scientists try to focus on activities that the bacteria or virus carries out that the host cell does not. Scientists often dissect the steps involved in infection and reproduction and ask whether they can block each one. For HIV the first interaction that scientists would hope to interrupt is the binding between the gp120 molecule on the virus and the CD4 receptor on the host cell. Some researchers have proposed using antibodies that recognize and bind gp120, in effect forming a cap that prevents access to CD4. The use of antibodies, however, begs the variability issue. To circumvent that, researchers have investigated the feasibility of using CD4 as a cap. They know that despite variations in gp120, it always recognizes CD4 and binds to it. Their hope was that soluble CD4 molecules would effectively tie up all of the viral gp120 molecules, and none would be available to bind to CD4 on cells. Unfortunately, this very clever strategy has not worked as anticipated. Through their studies the inventors of this strategy have discovered that to be effective, much higher quantities of CD4 must be administered than is practical. The next opportunity to interrupt the viral life cycle would be in the uncoating stage, but no drugs have been developed to prevent this yet. It is in the next stage, where the virus converts its genes into DNA from RNA, that drug developers have had the greatest success in controlling HIV. Drugs that interrupt viral DNA synthesis act to inhibit the enzyme called reverse transcriptase, which effects the RNA to DNA conversion. AZT is the oldest of the reverse transcriptase inhibitors and is most commonly given to people when they have fewer than 500 helper T-cells per microliter of blood. This drug and its close relatives dideoxycytosine (ddC) and dideoxyinosine (ddI) work through a trick they play on the reverse transcriptase enzyme. These drugs closely resemble the actual nucleic acid subunits that are strung together to make long chains of DNA, so the reverse transcriptase tries to incorporate them into the growing DNA molecule. However, a quirk in the chemistry of these nucleic acid analogs allows them to be added onto the DNA chain, but they themselves cannot form bonds with any incoming nucleic acid, meaning that they terminate DNA chain growth. The truncated pieces of viral DNA so produced are virtually useless and cannot direct the synthesis of new viral particles. AZT seems to slow the progression of AIDS once symptoms do appear and may reduce the number of opportunistic infections that

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A Positron Named Priscilla: Scientific Discovery at the Frontier afflict a person with AIDS. Early research suggested that AZT might delay the onset of disease in infected but healthy individuals, but more recent studies have hinted that it may offer no advantage to asymptomatic people. Although AZT is currently one of the best treatments available, it has many drawbacks. First, it is somewhat toxic. It also suppresses immune activity and causes some tissue damage. In addition, the virus seems to become resistant to AZT at an alarmingly quick rate—sometimes after only 1 year. For that reason physicians are now investigating the use of AZT in combination with or alternating with ddI and ddC, since all three drugs act in essentially the same way but have different sets of toxicities. These three drugs are the only ones approved so far by the Food and Drug Administration for use in treating AIDS. The ability of reverse transcriptase to mutate in such a way as to become resistant to AZT has inspired a new strategy for drug intervention. In laboratory studies, researchers exposed the virus to three different drugs targeted against reverse transcriptase, with the hope that the enzyme would become resistant to all three at the same time. These alterations would require the enzyme to change so much that it could no longer perform its DNA-synthesizing function. In theory, the virus would successfully resist the drugs but would become ineffectual in the process. This approach has produced disappointing laboratory results, and no one yet knows whether it will be effective or safe for humans, but it is an interesting example of how knowledge of the viral life cycle can lead to new and creative strategies for cutting that cycle short. In this case that means cleverly turning a viral defensive strategy against the virus itself. Another good target for therapeutic interventions is the integrase that incorporates viral DNA into the host chromosomes, but nothing has yet been developed that can do that. There is now a great deal of interest in agents that can inhibit gene activation by the Tat protein. While nothing truly promising has yet emerged, the hope is that when a Tat inhibitor is developed it will have the potential to block as much as 80 percent of HIV gene expression and possibly that much viral replication. If genes are expressed in the nucleus, however, a good target for drugs would be the Rev protein, which may help escort messenger RNA out of the nucleus and into the cytoplasm to be translated into protein. No Rev blockers have yet been developed. Once messenger RNA is in the cytoplasm its access to the protein-synthesizing machinery might be obstructed by the use of so-called antisense DNA. In this technology, strands of DNA can be manufactured

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A Positron Named Priscilla: Scientific Discovery at the Frontier to specifically recognize and bind to messenger RNA sequences that encode viral proteins. Once bound, the antisense DNA molecules make the messenger RNA molecules unreadable by the protein-synthesizing machinery. The potential use of antisense technology against HIV and other viruses has generated much excitement in the scientific community. Antisense is a highly specific strategy that holds the additional promise of circumventing viral latency. In fact, antisense technology is only useful when cells pass from latent infection to actively producing viral particles. In theory, all cells can take up the antisense molecules. As long as a cell remains latently infected, the antisense molecules would serve no purpose, but as soon as a latently infected cell starts expressing viral genes, the antisense molecules would effectively prohibit the manufacture of proteins from those genes. Without viral proteins, new viral particles cannot be made. So far, antisense technology has proved successful in the laboratory. Soon this technology will be used in human trials, where it is hoped, it will reduce the level of viral reproduction as well. Assuming a virus reaches the stage where it does manufacture proteins, the assembly and release of newly formed viral particles would constitute the last opportunity for drug intervention. Here researchers are testing the potency of a class of chemicals called interferons, which are normally secreted by virally infected cells. Although no one is sure of the mechanism, scientists have found that in the laboratory the specific compound called alpha-interferon inhibits HIV from appropriately assembling and budding out of the infected cell. The drug has been associated with some negative side effects, so its ultimate appropriateness for human use remains to be determined in clinical trials. In addition to drugs that interfere specifically with the viral life cycle, many new antibiotics and antimicrobial agents have been developed to fight the specific opportunistic infections that people with AIDS fall prey to (see Table 3.4). Despite the difficulty of finding a vaccine to prevent initial infection, physicians and researchers are optimistic that in the future AIDS patients will be healthier and more comfortable than in years past. In the years to come, AIDS may be a chronic disease, and people infected with HIV may be able to live with the infection for the rest of their lives. The new constellation of antiviral drugs and therapeutics directed against opportunistic infections may well mean that the relationship between infection and disease will change. A person who is seropositive for HIV may never develop AIDS or may remain disease free for many years.

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A Positron Named Priscilla: Scientific Discovery at the Frontier TABLE 3.4 Approved AIDS Medicines as of January 1, 1992 Drug name Manufacturer Indication Bactrim™+ trimethoprim and sulfamethoxazole Hoffmann-La Roche (Nutley, N.J.) PCP treatment Cytovene® ganciclovir (IV) Syntex (Palo Alto, Calif.) CMV retinitis Daraprim®+ pyrimethamine Burroughs Wellcome (Research Triangle Park, N.C.) Toxoplasmosis treatment Diflucan® fluconazole Pfizer (New York, N.Y.) Cryptococcal meningitis, candidiasis Foscavir® foscarnet sodium Astra Pharmaceutical (Westborough, Mass.) CMV retinitis Intron® A+ interferon alfa-2b (recombinant) Schering-Plough (Madison, N.J.) Kaposi's sarcoma NebuPent® aerosol pentamidine isethionate Fujisawa Pharmaceutical (Deerfield, Ill.) PCP prophylaxis Pentam® 300 IM&IV pentamidine isethionate Fujisawa Pharmaceutical PCP treatment PROCRIT®+ epoetin alfa Ortho Biotech (Raritan, N.J.) Anemia in Retrovir ®-treated HIV-infected patients Retrovir® zidovudine (AZT) Burroughs Wellcome HIV-positive asymptomatic and symptomatic (ARC, AIDS), pediatric and adult Roferon®-A+interferon alfa-2a, recombinant/ Roche Hoffmann-La Roche Kaposi's sarcoma Septra®+ trimethoprim and sulfamethoxazole Burroughs Wellcome PCP treatment VIDEX® didanosine (ddl) Bristol-Myers Squibb (New York, N.Y.) Treatment of adult and pediatric patients (over 6 months of age) with advanced HIV infection, who are intolerant or who have demonstrated significant clinical or immunologic deterioration during Retrovir® therapy Zovirax® acyclovir Burroughs Wellcome Herpes zoster/simplex   SOURCE: Pharmaceutical Manufacturers Association, Washington, D.C.

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A Positron Named Priscilla: Scientific Discovery at the Frontier Yet even though HIV-infected people may not develop symptoms of AIDS, one feature of infection will remain: They will still be able to transmit the virus to others. Here lies the final irony of the AIDS epidemic. While the world looks to science to end the spread of AIDS, the best solution is also the simplest and requires absolutely no technology. Scientists stress that no one ever needs to become infected again, if only people would take care not to engage in behaviors that place their tissues in contact with the body fluids of an infected person. However, if abstaining from these activities is not possible, the simple use of condoms during sexual activities would greatly reduce the number of new infections. Programs that encourage the use of clean needles by intravenous drug users also would drastically reduce the number of new infections. In short, the solution to the world AIDS crisis is to change human behavior. Therein lies the greatest hope and greatest tragedy for eradicating the new plague, AIDS. RECOMMENDED READING Benditt, J.M., and B. R. Jasny, eds. 1993. AIDS: The Unanswered Questions. Science, May 28, 1993. Volume 260, pp. 1253-1293. Mann, J., D. J. M. Tarantola, and T. W. Netter. 1992. AIDS in the World . Cambridge, Massachusetts: Harvard University Press. Shilts, R. 1987. And the Band Played On. New York: St. Martin's Press.