The Lake Casitas mouse � a model for the future of man

T.V. Rajan The Lake Casitas mouse – a model for the future of man? In 1982, I was a young Assistant Professor of Surgical Pathology at the Albert Einstein College of Medicine. What, you may ask, is a surgical pathologist and what might he do? Well, surgical pathologists are the quality control system in medicine. While most people may think that doctors make diagnoses by talking to or examining a patient, in many instances the diagnoses made during these office visits are only tentative and approximate. In many cases, doctors remove bits and pieces of tissue from various parts of the body (called biopsies) and send them to pathologists for examination under the microscope. The final diagnosis, the gold standard as it were, rests on the pathologist’s analysis of the tissue and confirmation of the clinical suspicion. While we pathologists do not have the kind of cachet that the highly sexy specialties of medicine such as vascular or neuro-surgery may have, the academic integrity of medicine lies in tissue diagnosis by pathologists. Most of the material that we examine on a day-to-day basis is relatively humdrum. In those days, one of the most common procedures that was performed in large city hospitals, such as the one at which I worked, was “termination of pregnancy”, a medical euphemism for induced abortion. In all of these cases, we had to confirm that the patients had indeed been pregnant and that the products of conception had in fact been evacuated. So, we would examine these TOPs as they were called under the microscope to make sure that the early fetal parts were recognizable in the blood clots that were removed from the patient’s uterus. This was important from a diagnostic and medico-legal perspective, but hardly challenging. Suddenly, almost hidden among the abundant ho-hum specimens, one stood out as rare, and therefore of great interest to us academic physicians. In principle, review of pathology with the pathologist is an important learning experience for every young doctor; in practice, most busy doctors with heavy clinical burdens seldom have the time to do this. This particular specimen was of sufficient rarity that almost every member of the infectious disease unit of the hospital visited the Department of Pathology to review the slides with us. It was a bronchial lavage fluid from a young man who had been previously healthy. Very suddenly, he had developed fever, cough and acute respiratory distress. MMWR, or Medical Mortality weekly reports, a publication sent to practicing physicians by the Centers for Disease control (CDC), had alerted the medical community that, over the last several weeks, a number of patients with similar histories were being brought to hospitals in the large cosmopolitan centers in the U.S., particularly Los Angeles, San Francisco and New York. MMWR also stressed that many of these patients presented with pneumonia caused by a very unusual etiologic agent called Pneumocystis carinii, an organism of dubious phylogenetic placement. As I mentioned earlier, clinical history and physical examination often lead to tentative diagnoses, and the clinical diagnosis in this patient had been PCP or Pneumocystis carinii pneumonia. But the P. carinii in lavage fluid 1 T.V. Rajan definitive diagnosis rested on the visualization by us pathologists of the causative organism. Everyone in the Department of Infectious Diseases wanted to be there when we looked at the specimen, so as to have a first hand look at this organism, which many had never seen before. Sure enough, the bronchial lavage fluid from this particular patient did indeed have the telltale signs of Pneumocystis carinii infection – dark, boat shaped organisms stained black by the silver stain used for their identification. We all stood around, excitedly discussing this new disease that was apparently sweeping across the country -- what it might signify, and why it was that these young homosexual men, who had never before had any symptoms, were coming down with this rapidly deteriorating course, typified by infections with this extraordinarily rare organism. We wondered about lifestyles, speculated about the rumor that was going around that the use of recreational drugs by these individuals was responsible for this outbreak. We had no data, just speculation. Over the next several months things changed dramatically and in ways that did not always make sense. Pneumocystis carinii, that uncommon infectious agent, peaked dramatically over the next few years to become one of the most common causes of pneumonia in young men. Then, prophylactic drugs were discovered that kept its incidence down until it was no longer the primary presenting symptom. Other bizarre manifestations such as Kaposi’s sarcoma, a tumor that we pathologists had known to be an indolent, slow growing, relatively non-threatening tumor in elderly men of Mediterranean and Jewish origins, suddenly showed up as aggressive, rapidly growing, lethal tumors in the same young homosexual men. Its incidence also peaked very quickly in the early 1980s and disappeared until it is seldom seen. Tuberculosis, a rare disease in this country, suddenly became rampant and alarmingly, resistant to most of the commonly used drugs to control these infections. In an extraordinary flurry of intellectual and scientific activity, the causative agent for this mysterious disease, human immunodeficiency virus-1 or HIV-1, was identified very quickly. We realized that the disease was a sexually transmitted retroviral infection. How best to deal with the global threat that this disease poses remains the subject of debate and confusion. What can be done to stem this pandemic that threatens human survival and well being in many parts of the world? The ravages of HIV/AIDS in Africa, particularly East Africa are well known. In countries such as Uganda and Kenya, large segments of the reproductively active age-group have disappeared, leaving young vulnerable children bereft of parental guidance or care. South Africa threatens to be another major disaster in the process of happening, whereas India and Thailand promise to be disasters in years to come. The extraordinarily efficacious drugs that have been discovered over the past decade for AIDS treatment (the so-called HAART or highly active antiretroviral therapy) are far too expensive for widespread use in impoverished countries where AIDS is rampant. Clearly the most logical approach would be prevention rather than cure. Spurred by this very reasonable rationale, some of the best minds in biology across the globe, have been working hard to define, design and deploy a vaccine that will not only prevent infection, but perhaps even cure the infection in established cases. Unfortunately, after the first flush of enthusiasm for this strategy, more recent and more mature reflection makes one wonder whether this is likely over the next several years or decades. The best paradigms we have for vaccines still remain those that are used for acute serious childhood viral infections. Most of these infections are caused by what we call lytic viruses. These viruses enter the host cells, undergo rapid multiplication, and cause death of the cells. If we can block this rapid 2 T.V. Rajan multiplication by antibodies that target the viruses and prevent them from entering fresh cells, we block infection. Unfortunately, viruses such as HIV-1 are far stealthier. Furthermore, HIV-1 has the extraordinary ability to enter one of the most important regulatory cells in the host’s antiinfection system. In so doing, it subverts the immune system to the point that a vaccine may pose technical and conceptual problems that we have still not yet faced. Despite the expenditure of enormous sums of money, there is no effective vaccine for HIV-1 in the horizon. Only one vaccine has even made it to Phase III trials, and it is unclear whether this will be the cure-all that we have been looking for. If there is no standard vaccine in the offing, is there another strategy? David Baltimore, the scientist who won a Nobel Prize (along with Howard Temin) for discovering the key enzyme that typifies retroviruses, has suggested an extraordinary strategy that he has called “intra-cellular immunization.” In order to understand this strategy, it is important to review some of the basic properties of viruses. Viruses lie at the interface between organisms that are truly alive versus those that are not. In order to be alive, an organism must possess two qualities – first, it must have the genetic information to make more copies of itself, and it must have the machinery to make these additional copies. Viruses possess the first, in the form of DNA or RNA genomes that encode all the information necessary to make additional copies of themselves. However, they lack the machinery to make copies of themselves, including the small molecular weight precursors, such as sugar, phosphate and nucleotides, and energy generating systems to synthesize large molecules such as DNA and RNA. They also lack the extraordinarily complicated machinery that a cell has in order to make new proteins, which are required to cover the viral genome in order to protect it from the harsh external environment. Since they lack this machinery, viruses are “obligate” intracellular parasites, and have to enter specific target cells in order to subvert the machinery of the host cell to replicate themselves. Implicit in this somewhat simplified formulation of the life of a virus are two requirements. The first step is that the virus must somehow recognize that it has encountered the appropriate cell in which it can replicate it, and secondly, that it must somehow enter the cell in order to use the cell’s machinery. How does the AIDS virus recognize the T cell and enter it? The virus is surrounded by a lipid membrane, and bears a major protein called gp160. HIV uses gp160 to identify target cells bearing two molecules on their surface in order to gain entry into them. One of these is a molecule called CD4, a protein that is present particularly on cells of the immune system, most notably T lymphocytes that orchestrate the immune system and macrophages that are the cells that usually ingest foreign particles and eliminate them from the body. Thus, HIV has subverted 3 T.V. Rajan the two most important cells in host defense for its own purposes. Another molecule that is used by HIV to gain entry into cells is one of the so-called chemokine receptors of which there are at least two that are relevant to HIV biology. Once gp160 binds to CD-4 and the chemokine receptors, the viral membrane fuses with the cell membrane and the virus gains entry into the cell cytoplasm. Here, an enzyme called reverse transcriptase, which the virus brings with it and which can copy RNA into DNA, converts the genome into a double stranded DNA, chemically the same molecule as our genome. This doublestranded DNA then enters the cell’s nucleus, where it become covalently and therefore permanently attached to the host’s genetic material. These DNA versions of retroviruses are called proviruses. The provirus uses host resources to copy the DNA back into RNA molecules (or original genomic form). The virus also subverts the host’s protein synthesizing machinery to make its own proteins, which encase the genomic RNA molecules and make more complete viral particles. The virus buds off from the cell without causing cell death immediately. Central to Dr. Baltimore’s idea of “intra-cellular immunization” is a phenomenon known as viral interference. From the ecological perspective of any parasite, this phenomenon makes good sense, in that any new, incoming parasite is potentially competing with an established infection. If the established infecting agent can prevent the incoming agent from establishing itself, the former has an advantage and does not have to compete for available resources. In the case of retroviruses, one possible mechanism would be if the endogenous virus were to cause the cell to make enough receptor binding protein (the “key” in our analogy above), that all the receptor molecules on the surface of the host cell (the “locks”) are occupied, so that no incoming virus can attach to them. This is shown schematically in figure 4. It is important to point out that the virus inside the cell does not have to be complete. It does not have to have all the genetic information to make more copies of itself. In other words, it could be “defective” in one or more ways. As long as it is able to make enough of the receptor binding protein, it can block incoming virus. In other words, it can behave like the proverbial dog in the manger – it is occupying the cell, it cannot use the cell because it is defective, but it still keeps competitors out! Is there evidence for such viral interference in the case of retroviruses and mammals? 4 T.V. Rajan One piece of evidence that this happens is that about 1% of the human genome is composed of DNA that is related to retroviruses. Most of these are very poor excuses for proviruses and are missing large chunks of genetic material. So, it is clear that they cannot make complete virion particles. How did these retroviruses come into our cells, when did they come in and why are they there? We don’t really know the answers to any of these questions but there is a lot of speculation about them. Based on our ability to investigate the timeframe of genetic change using mutation rates, researchers have determined that many of the retroviral elements in our DNA entered about 30 million years ago, about the time that we were separating from the great apes to become a unique species. It is unclear why there was such a huge burst of retroviral infection in our cells at that time. Relatively few viruses appear to have become stabilely integrated into our genome since then. Because they are missing so much genetic material, they cannot make infectious virus particles, but they can block retroviruses of the same family from reinfecting our cells. The working model is that when we were differentiating from the great apes, our species was subjected to invasion by many different retroviruses, many of which became stably incorporated into our genomes of partial proviruses. These now perform the important function of keeping us from getting infected by retroviruses to which they are closely related. Unfortunately, none of them is close enough to HIV-1 to interfere with that particular virus, and we as a species remain susceptible to it. As with any evolutionary story, there is an element of conjecture and plausibility, but seldom definitive proof. It is clearly impossible for us to return to 30 million years ago and ask exactly what the molecular events were that led to the incorporation of these defective viral genomes inside our own genome. It is in this context that the Lake Casitas mouse becomes an extraordinarily instructive example. The story begins with researchers from the University of California at Los Angeles who were interested in examining retroviral infections among wild type mice. They found a barn in a squab farm neat the town of Lake Casitas in California, where a colony of wild mice is flourishing, despite an epidemic of a particularly virulent form of a retrovirus. Analysis of the patterns of infection among these mice clearly documented that the virus was what is called an exogenous retrovirus, which was acquired by mice during breastfeeding on infected mothers. While the investigators were analyzing this population, it became clear to them that even within this very small ecological niche, there were mice that were clearly resistant to infection. Given the widespread presence of this virus, the resistance of this particular subset of mice was interesting in itself, potentially yielding clues about how to combat retroviral infection in a closed population. Investigators therefore analyzed the genomes of the resistant and susceptible populations of mice using standard genetic techniques, and quickly determined that there was a stable, heritable genetic locus in the infection resistant mice that was not present in the infection susceptible mice. One of the great advantages of modern molecular genetics is that once a genetic locus has been identified, it is far easier to determine its structure and function today than it was during the time of Mendel. Molecular cloning of this particular locus revealed that the locus consisted of a highly mutated form of the DNA copy of the retrovirus. The data clearly showed that within this colony of mice, one or more founders had become viremic to the point that the viruses were infecting tissues other than the cells of the central nervous system. Presumably in one or more such mice, the virus had infected the germ cells and caused a stable integration of a mutated form of the virus. This virus was then being passed along to the progeny of such mice, just like any other genetic locus that is part of the mouse genome. Because of the fact that this virus was highly mutated, it was not producing viral particles and 5 T.V. Rajan causing disease from within; instead, it was producing enough viral envelope protein, the “key” that binds to the cell surface “lock” on target cells, that the receptor was being occupied and preventing exogenous virus from entering susceptible target cells. Thus even though these mice were becoming, presumably, infected by the virus that was present in the environment of the barn, the fact that they had inherited a defective copy of the virus from their parent or parents made them resistant to viral infection and therefore protected from the ravages of the disease. Thus, the Lake Casitas mouse offers an extraordinary example of an ongoing evolutionary process that describes how it is that in a population at large viral infections may be dealt with through evolutionary forces. Given the efficacy with which this process works, a number of investigators have begun to question whether a similar approach might be a viable strategy for dealing with the global threat posed by HIV-1. In thinking about any vaccine, most scientists have come to classify them into two overlapping strategies. The first is the so-called prophylactic vaccine, one that is delivered either to populations at large or to susceptible or potentially susceptible individuals, and to protect them from the possibility of becoming infected. The alternative strategy is to use a vaccine to cure infections in already infected individuals. Most of the existing vaccines, particularly the ones that are widely known among the public, belong to the first group and have been widely deployed particularly among children in order to prevent them from getting infected with such viruses as chickenpox, mumps, measles, German measles and other childhood viral infections. These are relatively few examples of the so-called curative vaccines. It does appear, however, that in the case if HIV the curative possibilities of these highly debilitating viruses are much more appealing than the prophylactic version. The reason for this is that it appears that even highly debilitated viruses somehow become capable of producing virulent viral particles that can cause disease. This is clearly ethically unacceptable for deployment among large populations, because this would imply that innocent individuals could become infected with a disease that might not strike them. An example of this comes from the work of DeRosiers and his colleagues at the New England Primate Center. It was announced to great fanfare that seriously debilitated copies of the a simian retrovirus (that causes a disease very similar to AIDS in monkeys) could provide prophylactic immunity to vaccinated monkeys. Unlike the many subunit vaccines that have been tried thus far, this whole attenuated viral strategy appeared to give the infected chimps protection far more robust and far more complete than had ever been achieved. Unfortunately, however, even though these viruses had been profoundly debilitated by DeRosiers and his colleagues by deleting segments of multiple genes, it became clear that with the passage of time these viruses were somehow reverting to become complete virus and were producing infectious particles in some of the vaccinated animals. Similar results have been found by other investigators as well. This situation clearly poses an ethical dilemma. The principle of prophylactic vaccination is to avoid or at least ensure the absence of infection in those that volunteer to receive the vaccine. If we cannot guarantee that they will not be infected, ethical considerations would prevent the deployment of such vaccines in large populations. Is there scope then for such a vaccine? The answer appears to come from the work of Kohn and his collaborators at the University of California’s Children’s Hospital Center. In order to understand their work it is important to review briefly the mechanism by which the cells in the blood are generated within the body. Inside the bone marrow in your body are cells known as stem cells that generate, through the process of repeated multiplication and the expression of specific functions, every 6 T.V. Rajan type of cell that is present in blood including red cells, white cells and platelets. These cells are characterized by the expression on their surfaces of a molecule called CD34. If one were to irradiate an individual so as to kill his bone marrow, he or she would die because of the lack of white blood cells to kill infectious agents. However if such an individual were to be given CD34+ cells, his bone marrow would be rapidly repopulated by these cells which would then generate red and white cells to ensure that he does not die. The strategy of defective viral infection of these CD34 cells provides an elegant possible solution to treating individuals who are currently infected with the AIDS virus. The approach would be to remove CD34 expressing stem cells from the bone marrow infected patients and infecting these CD34 cells with defective copies of HIV. If these CD34 are then put back into the AIDS patient, they would repopulate the bone marrow and give rise to T cells that now contain within them this defective copy as part of their genome. This “endogenous” copy of the retrovirus would then start making the viral protein but because it is defective it would not be able to give birth to complete viral particles. These viral proteins would then interact with cell surface receptors for the genuine HIV virus so that these T cells would not be infected even though the environment of the patient contains infectious HIV particles. In other words, within the ecological niche of the AIDS patient, we have a scenario that is very similar to that in the Lake Casitas mouse. Just as there are individual mice that cannot be infected by the virus in the barn environment because they have inherited a defective copy of the virus, we now would have in the ecology of this patient individual CD4 expressing T lymphocytes that cannot be infected by the virus present in the environment, because of the inheritance of defective retrovirus from within. Thus, even though the patient is currently infected with the AIDS virus, we can generate functional CD4 expressing T lymphocytes and restore immune function and improve longevity of the patient. Tampering with the genomes of people clearly creates profound ethical dilemmas. However in this instance we are changing the somatic cells of the individual, not the germ cells. It is true that this individual would have, in his or her bone marrow, cells that contain, as part of the genetic material, an introduced defective viral copy, this virus is not capable of replication. Therefore it cannot be transmitted to the next generation. This would therefore reduce the ethical dilemma posed by many genetic engineering approaches. How about the fact that these “defective” viruses seem to be capable of generating intact viruses as we noted earlier? This too does not create a problem in a patient who is already infected with HIV. Even if the proviruses somehow correct themselves and start making intact HIV, this reversion will not add to the virus pool already existing in the patient’s circulation. Whereas this type of vaccine is an ethical problem for individuals who are uninfected, it does not pose the same danger for individuals who are already infected. Thus it is intriguing that a colony of mice in an obscure barn in California could well provide a model with which we could approach the management of large numbers of people with the AIDS virus infection in countries that could ill afford HAART therapy. Technical limitations at the moment make it unlikely that this therapy will become widely available or used in the near future. While in principle CD34+ cells can give rise to every leukocyte and red blood cell in circulation, in practice it has been difficult to get good repopulation thus far. It is likely that this represents a technical problem that is not insurmountable. There may come a time when there are individuals in third world countries that would otherwise die from their AIDS virus infection were it not for the lessons learned by retroviral biologists working in models that seem to be far removed from the so-called “real life situations” that involve real patients in real countries. 7