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Page 96 Appendix D Vaccination Vaccination is the deliberate immunization of an organism against infection by a disease agent. It can also mean the immunization of a person against any agent capable of provoking an immune response. The agent provoking the response can be an infectious organism, but it can also be a medium-sized molecule (i.e., a protein toxin) or part of a protein from one’s own body (e.g., stimulating an immune response to a tumor). Anticipated advances in cell biology, immunology, molecular genetics, genomics, and cellular immunity will greatly accelerate the production of cheap, safe, effective vaccines. Vaccines prepare the immune system to recognize and attack invaders or antigens. Once an organism has been vaccinated, it becomes immune because it contains populations of cells carrying molecules on their surfaces that recognize particular antigen molecules produced by, or part of, an infectious agent. Antigens are frequently, but not always, proteins encoded by the genome of the infectious agent; they can also be other molecules, such as complex carbohydrates. IMMUNE RESPONSE The technology of vaccination was worked out considerably before any of the underlying science was understood. In the late nineteenth century, the observation that people who had been infected or exposed to a disease agent were subsequently immune to it was extended by rigorous experimentation. The panoply of responses to an invader became known as the immune response. The fact that the immune response is faster and stronger on reinfection than on first infection, meaning that the organism “remembers” that it has once been infected and remembers how to deal with it, became known as immunological memory. The organismic system responsible for the immune response became known as the immune system. The different types of (white) cells that orchestrate and execute this complex behavior circulate freely in the blood, where they are relatively easy to get at; for this and other reasons, advances in understanding the immune response in the twentieth century were breathtaking. The exponential increase in scientific knowledge is playing out in sophisticated methods (tactics) of exploiting the immune system to meet specific goals. In the textbook picture, the immune system is divided into two arms, one that uses antibodies to recognize and help destroy intruders (also called B-cell arm, humoral immunity, and serum immunity) and one that uses cells (also called T-cell arm and cell-based immunity). A stripped down version of the canonical picture of the natural history of a “typical” immune response is described below. An invader, let us say a bacterium or a virus, replicates, let us say in the blood (or, if a virus, in the cells of the blood). The invading organism is made up of molecules, lipids, nucleic acids (e.g., DNA) and proteins. The molecules, and parts of molecules, that are recognizable by the immune system are referred to as antigens. B-Cell Arm Some of the invading organisms circulate in the blood and lymph. In the spleen, antigens on some of the viruses match (are recognized by) antibodies on the surface of particular B-cells. When cells that bear these antibodies start dividing, they create a clone of cells descended from the founder cell that recognized the antigen (clonal selection). Many of these daughter cells begin to make mutant antibodies, some of which bind more tightly to the antigen. Those that do proliferate more. Thus, the longer the antigen is around, the more cells make antibodies that bind it strongly. Later, cells that carry the tight-binding antibodies on their surfaces rewire themselves internally so that the antibodies are secreted into the blood and lymph. If the invader is a virus, circulating antibodies destroy the virus by binding to it and gunking it up into large complexes (immune complexes) that are cleared from the blood. If the invader is a bacterium, complex formation can happen as
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Page 97well, but, in addition, the binding of the antibody to the large bacterium triggers a complex series of events, all caused by proteins acting on other proteins (the complement cascade). The call-down of the complement cascade results in the bacterium being punched full of holes, which, as ions leak in and leak out, kills it. After the infection is over, a small population of B-cells rewires itself to express on its surface the antibodies that worked best. If the invader returns, these cells (memory cells) proliferate immediately. On the second pass, therefore, the process does not start anew but begins at the end of the cycle. T-Cell Arm Some of the invading bacterial and viral particles are ingested by antigen-presenting cells (APCs), such as macrophages and dendritic cells, which in turn chew up the viral proteins and display pieces of them on their surfaces. The APCs collide with other cells, including one flavor of T-cells, called T-helpers (actually Ths, of the Th1 subclass, so Th1 cells). Some Th1s have, on their surfaces, molecules that happen to bind to one or another piece of chewed up viral protein (the antigen). The helpers make soluble proteins (e.g., interleukin 2, or IL-2) that stimulate the other main class of T-cells, called killers (Tk), also known as cytotoxic T-cells (Tc), to divide. If a cell is infected with a virus, some of the proteins it makes are encoded by the virus. The cell displays pieces of these viral proteins (as it displays pieces of its own proteins) nestled in a cleft on a particular kind of molecule called HLA class I. Tk cells have on their surfaces molecules called T-cell receptors (TCRs). TCRs on different Tk cells come in many different shapes, and some Tks have TCRs that recognize and bind to the complex between HLA class I and viral proteins found on the surface of the virally infected cell. When a Tk has bound to a cell, it is said to be antigen activated. In the presence of IL-2 from the Th1 cells, these antigen-activated Tk cells proliferate and turn into active killers. When an active killer runs into a foreign cell with HLA on its surface that it recognizes, it binds to it, punches holes in it, and kills it. When the immune response is going well, the killer can recognize the virally infected cell and destroy it before progeny viruses are produced. Antigen-activated Tks tend to persist. On reinfection, in the presence of IL-2, they proliferate again, and the reponse picks up at the end of this stage of the process. After vaccination, the organism that has been vaccinated is said to be immune; that is, it cannot be easily reinfected by the disease agent. Immunity means it now contains populations of cells that carry on their surfaces molecules that recognize particular molecules (antigens) that are made by, and are part of, the infectious agent and that are recognized by the immune system. That is, antigens are defined operationally as those molecules or parts of molecules to which the immune system responds. Antigens are frequently but not always proteins encoded by the genome of the infectious agent; they can also be other molecules, such as complex carbohydrates. It is perfectly appropriate to think of the immune response as an amazingly sophisticated identification-of-friend-or-foe (IFF) defense mechanism. In this view, the immune system represents a paradigm of a distributed network that can perform a sophisticated task (recognition of an intruder, decision making, execution). The response is robust, redundant, and nonhierarchical. Communications among components of the immune system are of two different, low-bandwidth types: (1) cell-cell touching, which can convey pieces of molecules (high-semantic content) and (2) secretion of proteins (~100 different proteins in all), which are received by cells if the receiver cell has the right receptor and if concentration of the secreted protein is high enough. Although local cells may be more affected, the communication is nondirectional spatially; that is, components of the system exist in no known, or fixed, orientation or relationship to one another. This combination of high and low bandwith, slow signaling, decentralized decision making, and timely, accurate response is unparalleled in the design and manufacturing world. VACCINE DEVELOPMENT AND PRODUCTION Recent developments in cell biology, immunology, molecular genetics, and genomics relevant to the Army are likely to lead to the development of less complex vaccine products (e.g., recombinant viral proteins or DNA encoding for these proteins), the generation of protective immune responses against a wide range of pathogens, and the creation of well-characterized products manufactured by robust, fast, inexpensive process technologies. Generally, six types of vaccines are used today: 1. killed infectious organisms (inactivated vaccines) 2. naturally occurring infectious organisms that are closely related to the pathogen (e.g., the cowpox virus used in vaccinations against smallpox is closely related to smallpox but does not cause the disease in humans) 3. live infectious organisms that have been treated, or mutated, to be less virulent (live attenuated vaccines) 4. subunit vaccines 5. cell-based and virus-based vaccines 6. DNA vaccines The impacts of developments in biotechnology on types 3, 4, 5, and 6 are described below. The impact on adjuvants, substances used in vaccines to increase the strength of an immune response, are also described. The final section is a discussion of how recent developments in biotechnology could open the way to new ways of conferring immunity.
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Page 98 Live Attenuated Vaccines Selected attenuated vaccines are conventionally generated by growing a bacterium or virus over many generations in a host different from the organism to be vaccinated. Because growth in a different host organism exposes the pathogen to a different selective environment, the pathogen undergoes a number of mutations that adapt it for the new host and make it less fit in its normal host. For example, to generate a virus against influenza, the new flu strain is grown for many generations in chicken eggs. Whatever helps the virus grow best in chicken eggs is not the same as whatever helps it grow best in humans, so it is less fit in humans and less able to cause disease in the original host. These debilitated or weakened mutants are selected for the vaccine. Recently, at least for viruses, this classical approach to selecting mutant viruses has been supplemented by the deliberate introduction of random or directed mutations. Selecting viruses that are, for example, temperature-sensitive results in viruses that are less able to grow well in the host. Directed mutagenesis (mutation development) of genes in viruses that are antigenic is another widely used technique. A gene that is useful but not necessary for growth, for example, can be removed and tested to see if infection with the mutant virus confers immunity. These obvious molecular tactics could be extended to bacterial pathogens. Subunit Vaccines In the last century, biology was focused on individual molecules responsible for disease and immunity. Today, the vaccine industry has largely moved beyond the use of killed or live attenuated whole organisms to vaccines that contain key molecules sufficient to confer immunity. Genomics is moving the science and practice of vaccination down to the level of the genes that confer immunity. Most modern vaccines are subunit vaccines, that is, vaccines that contain one or more molecules or parts of molecules that carry the immunological properties of an organism, which in turn can elicit the immune response. Generically, the process of generating a subunit vaccine for an organism involves a survey of organisms that had been infected with the infectious agent and recovered to determine which antigens provoked an immune response. Those antigens, typically proteins, are then manufactured, typically as recombinant proteins expressed in bacteria, yeast, or cultured animal cells. The recombinant proteins are typically mixed with an adjuvant and injected into test animals to determine if they confer immunity. Based on these animal studies, the mix of antigens and adjuvants can be fine tuned. Eventually, trials are conducted in humans. Recombinant DNA technology and immunology were successfully used in the search for the surface antigen of an invading organism and the development of a recombinant vaccine for hepatitis B. The tools and methodologies for this concept are well proven and could be easily extended to other systems. Moving from flu vaccine in chicken eggs or vaccinia from cow pustules, to cultures of pure organisms in vitro to recombinant proteins from those organisms, leads to well defined and pure vaccines. Less complex vaccine products, such as recombinant proteins and DNA, are generally easier to manufacture and characterize. Therefore, reproducible, consistent processes might be developed for producing them at reasonable cost and on reasonable schedules. For example, the United States has far more cell culture capacity for producing most recombinant protein and plasmid DNA products (biosafety level GLSP/BL-1 cell-culture capacity) than capacity for producing vaccines from pathogenic infectious agents (BL-2+ cell culture capacity). Given the complexities associated with supplying vaccines for the Army, manufacturing strategy should be considered at an early stage in their development. Genomic techniques promise to simplify the task of identifying candidate antigenic molecules. Antigenic proteins tend to be on the surface of the bacterium, and the set of proteins expressed on the surface when growing inside cells is likely to contain almost all of the proteins made by the bacterium that causes the immune response. Similarly, antigens are likely to be found in the set of proteins secreted by the pathogen and among the genes that are transcribed in the messenger RNA during the course of an infection. Consider a bacterium that grows in human cells, for example. Inspection of its genome sequence is now sufficient to identify the protein molecules encoded by its genome that might be expressed on its surface. The subset of those proteins that are expressed when the bacterium grows inside cells can be identified by mRNA expression analysis, which should give the most likely antigen candidates. As always, improvements in underlying technologies will accelerate these analyses. For example, sequencing of single strands of DNA through nanopores may make possible the sequencing of entire bacterial genomes at much higher speeds. Cell-Based and Virus-Based Vaccines In cell-based vaccines, nondisease-causing cells or viruses are genetically altered to display antigenic molecules, typically antigenic proteins, derived from an infectious organism. The displaying cells can, for example, be beneficial bacteria that usually inhabit the respiratory tract or gut, or even somewhat pathogenic bacteria that cause a mild infection and displace existing flora for a few days. These altered cells are used as the vaccine to elicit the immune response. Despite some research in this area, much of which was funded by the Defense Advanced Research Projects Agency (DARPA) Unconventional Pathogens Countermeasures Program, no cell-based vaccines have been approved for use against infectious diseases. However, they are used in cancer therapy for myelomas. Nowadays it is reasonably
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Page 99common to take tumor cells from a patient, grow them in vitro, and reinfuse them into the patient in an attempt to cause the immune system to react against the cancer. The effectiveness of these vaccines can be increased by introducing into the tumor cells DNA that directs the synthesis of proteins that increase the antigenicity of the cancer cell. This approach has been very successful against adult-onset leukemia and lymphoma, curing 50 percent to 80 percent of patients who would otherwise have died. However, trials have been limited to dozens of persons. Another promising approach is the use of viral vectors (engineered viruses) to deliver antigens that confer immunity against multiple infectious diseases in one vaccine. For example, consider vaccinia, the cowpox virus used to immunize against smallpox. Vaccinia, a double-stranded DNA virus with a large genome, contains enough dispensable DNA in principle to direct the synthesis of numerous foreign proteins. That is, pieces of vaccinia DNA can be cut out and replaced with other DNA that encodes other antigens. Vaccinia derivatives were produced during the 1980s that could direct the synthesis of foreign proteins based on this principle; one could imagine using vaccinia or other vectors that carry multiple antigens to develop a single vaccine that would confer immunity against multiple infections at a very low cost per dose (e.g., $0.25). DNA Vaccines DNA vaccines are another promising new type of vaccination. Because DNA can be manipulated much more easily than proteins or living organisms, the development of DNA vaccines raises the possibility that new vaccines, or vaccines against new organisms, could be generated and distributed quickly—within weeks of identifying a pathogenic organism. In one version of this approach (developed at the University of Texas Southwestern Medical Center in Dallas), fragments of DNA that direct the synthesis of protein antigens specific to that organism are generated by polymerase chain reaction (PCR) from the genome of the organism. The assembled piece of DNA is mixed with other DNA constructs that direct the synthesis of cytokines or proteins (e.g., interleukin-12) that stimulate the immune response. Gold spheres with slight surface roughness are mixed with the DNA, and the spheres are shot (using a gene gun) into the skin of the animal being immunized. Some of the DNA is taken up by dendritic cells in the skin and presented to the immune system, which initiates the process leading to an immune response. The ultimate promise of this technology is the deconvolution of a pool of genes in a whole-genome, shotgun approach in a few weeks, as opposed to years. To identify the particular genes that would confer immunity, the researcher could take all of the potential antigen-encoding DNA, break it into pieces, amplify it using PCR, express it in mice, and find the products that render the mouse immune to infection. (The limitation is that mice are not the same as people and are not susceptible to all of the same diseases.) Conceptually, the information-processing part of generating DNA vaccines is identical to the technique used for subunit vaccines: the antigenic molecules (here, exclusively proteins) are identified and tested in animals. The pieces of DNA that go into the vaccine must then be specified. But because it is so much easier to manipulate DNA than produce numerous different recombinant proteins, it becomes cost effective to pursue tactics such as immunizing different animals with all possible different genes or combinations of genes (pooling approaches), identifying which animals become immune, and combining the genes that work in a single vaccine. DARPA has invested heavily in this technology. In fact, the United States’ rudimentary, experimental surge capability to make vaccines is almost entirely attributable to the DOD research. DNA vaccines are now being pursued by many U.S. biotechnology and pharmaceutical companies. The Army should keep abreast of this research. ADJUVANTS An adjuvant is a substance that increases the immunogenicity of an antigen. Adjuvants work by, for example, increasing the number of APCs (antigen-presenting cells) that migrate to the site of the vaccination or by emulsifying the antigenic proteins in the vaccine preparation so that they are partly unfolded and can be more easily recognized and ingested by the APCs. In some ways, the use of adjuvants is a century-old technology. However, recent advances in the understanding of the immune system have brought to the attention of vaccine makers molecules (e.g., interleukin-12) that can stimulate the immune system by well understood mechanisms. The Army’s vaccine program has a long-standing interest in adjuvants and is already following these developments closely. IMMUNITY BY OTHER MEANS Two other means of conferring immunity could be affected by emerging biotechnologies: passive immunization and innate immunity. Passive Immunization An injection of antibodies (IgG) from an immune individual confers a transient, IgG-based immunity to the disease agent as long as those antibodies continue to circulate in the blood of the person receiving the injection. Because there are no memory cells in the immunized individual, protection lasts only as long as the injected antibodies continue to circulate (typically, weeks). Therefore, the immunity conferred by this technique is called passive immunity. At the
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Page 100start of the twentieth century, sera that confer passive immunity to infectious diseases, such as diphtheria, were generated by injecting antigens into horses and purifying the IgG fraction from their serum. This technique is still sometimes used. For example, a person traveling to a country where a disease, such as hepatitis A, is endemic might receive an injection of gamma globulin made from a human who has recovered from the disease. With advances in understanding, new techniques are being developed. A number of means are now used to isolate genes that encode human (or humanized) antibodies against any antigen. Once the gene has been isolated, the antibodies can be produced in vitro, in cell culture, or in milk, eggs, or even plants. Both for diagnosis and for prophylaxis (i.e., vaccination), the United States should develop a surge capacity to produce antibodies. DARPA has at least considered funding such work, and the Army should support it. Stimulating Innate Immunity The complex B-cell and T-cell arms of the immune system enable the organism to become immune to an infectious agent after its immune system is exposed to the agent. Organisms also possess innate immunity, which enables them to respond to infectious bacteria and viruses to which they have not been previously exposed. Some mechanisms of innate immunity predate the modern immune system. For example, cells have receptors for types of molecules found on bacterial surfaces, and when those receptors are bound, the cell becomes more resistant to infection. Much more recently, vertebrates have evolved other pathways. For example, interferons cause most cells to initiate responses that make it more difficult for viruses to grow in them. There are also “natural killer” (NK) cells that can rapidly mobilize and attack foreign cells without having been educated as to the nature of the foreign antigens. The complex of cytokines that choreographs and directs the NK response is increasingly well understood, but the mechanism(s) by which NKs recognize cells as foreign is not. In the next 25 years, more will certainly be learned about the molecular nature of these phenomena; eventually it will be possible to manipulate these mechanisms to confer immunity or to block the progress of an infection once it has started. These techniques are likely to be too experimental to be pursued commercially. DARPA is now one of the lead agencies funding this promising research, and the Army should follow developments closely. GLOBAL IMPACT OF ARMYDEVELOPMENTS The Army wishes to be able to protect its forces against disease no matter where those forces may be deployed. By contrast, the pharmaceutical industry requires the projection of peak annual sales on the scale of hundreds of millions of dollars per year before deciding that a drug will be sufficiently profitable to justify its commercialization. The markets that can bear this expense are the approximately one billion people of the affluent areas of Europe, Japan, East Asia, and North and South America. Because commercial BOX D-1 Malaria Malaria is a global public health problem. A staggering 40 percent of the world population lives in areas where malaria is transmitted, resulting annually in an estimated 280 million to 1 billion cases, and 1 to 3 million deaths (WHO, 1999). The most severely affected populations are young children and pregnant women and infants. The threat of malaria is not restricted to people living in malariaendemic areas. Military personnel, State Department personnel, and travelers who visit these areas are also at risk. Infection with Plasmodium falciparum, the most virulent human-infective parasite, can be fatal to nonimmune individuals. In addition, in many areas the parasite is becoming resistant to the current prophylactic antimalarial drugs. The development of new antimalarial drugs is lagging far behind the development and spread of resistant parasites. Mosquitoes, the insect vector for malaria, are also developing resistance to insecticides. Even if effective, insecticides are too costly to be a viable control measure in malaria-burdened nations. An obvious solution for the failure of antimalarial drugs and the difficulties inherent in large-scale mosquito control is the development of an inexpensive vaccine that could be used to protect both travelers and indigenous people in malarious areas. In recent years, several promising vaccine candidates have emerged, but progress has been slow. A major impediment has been lack of sufficient funding to support malaria research. In 1999, for example, the National Institutes of Health, the leading funding body for research in infectious diseases in the world, allocated $24 million for extramural research on malaria. This level of funding falls far short of the estimated $1.6 billion some believe is necessary (CID, 2000). Research should be focused on an in-depth investigation of the complex relationships between the malarial parasite, the human host and the mosquito vector, and the nature of the immune response that must be elicited by a vaccine. The natural development of immunity to malaria in exposed people, such as children who survive multiple infections in their first few years of life, and the alteration of immunity experienced by pregnant women, should be explored. How these immunological changes will affect the delivery and efficacy of a malaria vaccine will also have to be investigated. Malaria is not only a problem of developing nations. It also has important implications for the continued development and prosperity of all nations. Concerted global efforts, starting with vigorous research and culminating with the delivery of effective, sustainable, affordable interventions for everyone at risk would have large-scale benefits. Source: Moore, 2000.
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Page 101 small-molecule drug development has been focused on specific targets, it is unlikely that therapeutics will emerge for unrelated diseases. Even research on diseases common to rich and poor countries typically target the strains of the disease found in rich countries. Since Army forces are far more likely to be deployed to poorer countries, the Army should identify ongoing developments that it could use to leverage future development of therapeutics against the infectious diseases found in poorer countries. For years, the Army has been one of the main promoters of the development of drugs against diseases endemic to poor countries, including malaria, which is responsible for 1 million to 3 million deaths annually (see Box D-1). In the future, the Army could have a considerable impact on the treatment of bacterial and protozoan parasitic diseases and viral infections that are prevalent in the relatively undeveloped areas of the world. By 2020, an estimated 7 billion to 8 billion people will live in less affluent areas of the world. Because it must be prepared to deploy forces in these areas, the Army should continue to remain closely involved in the development of therapeutics that could not only protect U.S. forces, but also contribute to world health.
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