Acute Respiratory Diseases in Children: A Research Grant Area of the Board on Science and Technology for International Development

Acute respiratory infections (ARI) are still the world's leading killer of children. Through its research grants program supported by the U.S. Agency for International Development, BOSTID's Committee on Research Grants is funding etiologic studies in 12 countries. They will provide a critical first step toward developing effective treatment protocols and, possibly, a vaccine. An intensive effort is presently under way to analyze and interpret the substantial data that have been gathered.

In drawing conclusions from this data, grantees are grappling with such problems as the finding that the incident rate of ARI in children is no greater in the Third World than in the United States, yet the mortality rate is 20 times higher in developing countries. The available data cannot answer whether the important factors are malnutrition, other infections, or unrecognized coinfections. It has been agreed that future studies will be designed to answer these critical questions, but that the results of the current etiologic research to determine the principal causal agents are a prerequisite for an attack on the pathogenesis problem.

At a 1986 meeting of the grantees, ARI researchers reported on a rapidly expanding body of data. Respiratory syncytial virus is the virus most closely associated with severe ARIs, but other viruses are also seen. Speculation centered on the idea that the viral infection leads to severe bacterial infections in the worst cases.

Present State of Biotechnological Research on Vaccines

Before describing recent developments in vaccine research, Warren pointed out that biotechnology is "a very young discipline in terms of the I practical outcomes of what's been done," yet it has already produced an I excellent and very powerful vaccine. The hepatitis B vaccine has been , approved by the U.S. Food and Drug Administration and is licensed for use. Hepatitis B is a major cause of liver cancer throughout East Asia and Africa.

As of April 1986 the U.S. Army had 42 new vaccines under development; the National Institutes of Health, 28 vaccines; and the Rockefeller Foundation, 6 vaccines. As for priorities for vaccine development, the Institute of Medicine compiled its own list of priorities based on a combination of the importance of the disease and the state of research that is likely to lead to a vaccine in a relatively short time.¹ The five priorities listed for the United States were hepatitis B, respiratory syncytial virus (which causes most of the croup in children in the developed world and is a major killer in the developing world), Hemophilus influenzae Type B, influenza virus, and chicken pox. Priorities for the developing countries were pneumo-coccal pneumonia, malaria, rotavirus (one of the major causes of diarrhea and one of the major killers of infants in the first year of life in the developing countries), typhoid bacillus, and shigella (bacillary dysentery).

The polio vaccines have made news again. The protective antigen from polio vaccine 3 has been genetically engineered into polio vaccine 1, resulting in one vaccine that protects against polio viruses 1 and 3. Most likely polio vaccine 2 will soon be combined with the other two into one vaccine that immunizes against all three strains of the disease.

As for the two childhood diseases pertussis and measles, a new acellular vaccine has been developed for pertussis in Japan that is much less toxic. It is being tested in Scandinavia. And the newest measles vaccine, Edmonston-Zagreb, is being tested for administration to children as early as four months of age.

In another development, genetic engineers have cloned the entire gene structure of Mycobacterium tuberculosis, of respiratory syncytial virus, and of human immunodeficiency virus (HlV), the cause of acquired immune deficiency syndrome (AIDS).

Challenge studies recently undertaken with mosquitoes carrying living malaria have shown that the first synthetic malaria vaccine in human trials offers hope for protection against malaria. According to Warren, this is indeed a major breakthrough and may signal the introduction of a practical malaria vaccine.

The new typhoid vaccines are living typhoid bacteria with a defective biochemistry. Thus, when given to children the living bacterium penetrates the intestinal wall, and because it is missing certain crucial enzymes, it slowly dies in the tissues. Immunization results from the inability of the living bacillus to live within human tissues. Other genes can be inserted into the Salmonella bacterium so that theoretically one oral dose of the typhoid vaccine can protect against a whole range of intestinal infections.

The gene deletion technique - deleting the part of the gene responsible for causing disease - has been used to delete the cholera toxin from cholera bacillus. This toxin causes severe diarrhea.

The new and very powerful technique of gene insertion is being used to turn the vaccinia vaccine (the old smallpox vaccine) into a vaccine that can protect against a large number of different diseases. As many as 20 different genes can be inserted into this vaccine, which is then scratched into the skin in a simple procedure. The twenty-fret disease is smallpox itself, which fortunately is no longer needed because this disease has been eradicated worldwide.

Even antifertility vaccines have been developed, and the first was tested in India several years ago. The second is being tested now in Australia.

Mosquito Vector Field Studies: A Research Grant Area of the Board on Science and Technology for International Development

With the support of the Office of the Science Advisor, U.S. Agency for International Development, BOSTID's Committee on Research Grants is funding a small number of research projects in developing country institutions that focus on the population ecology of mosquito vectors in relation to the epidemiology and control of human diseases.

These investigations are seeking to characterize vector habitats; quantify vector abundance and seasonal or year-to-year variations and survivorship; document the interaction of vectors, vertebrate hosts, and pathogens; and identify the factors affecting vector abundance (including survival).

Despite research advances in mosquito vector studies, the disease problems have worsened. Malaria is spreading to areas where it had not recently been a problem, and resistance of mosquitos to insecticides and of parasites to medication is spreading at an alarming rate. Several BOSTID researchers have revealed issues that are more serious than anticipated, involving genetic-linked geographic diversity (implying that experience with control of an anopheline species in one place may not be valid for the same species in another) and interaction of vector species with human habitats. Some new technologies have proved successful, however, in the battle against malaria - for example, enzyme-linked immunosorbent assays (ELISA) for detecting infected mosquitoes and DNA probes for identifying hard-to-distinquish sibling species.

Finally, the latest finding in vectors is the shuttle plasmid, a phage that goes into mycobacteria as well as a plasmid that goes into E. coli. According to Barry Bloom, author of "Introduction of Foreign DNA into Mycobacteria Using a Shuttle Plasmid" (Nature, June 11, 1987), "it may be possible to develop mycobacteria into useful multi-vaccine vehicles."

One almost unbelievable problem faced at the moment, noted Warren, is the plethora of prototype vaccines for some diseases. At least six different rotavirus vaccines are now being tested in children, and at least eight different vaccines for controlling schistosomiasis are in the laboratory.

Warren ended his presentation by calling attention to the efforts being made by UNICEF, WHO, USAID, and others to make universal childhood immunization a reality. In a big step toward this goal, China has agreed to raise its childhood immunization rate from 50 percent to 100 percent, and India has dedicated to Indira Gandhi a major campaign designed to immunize all its children by 1990.

"The feedback loop has been established," concluded Warren. "The children of the world are being immunized and if the power of biotechnology to produce new and better vaccines is fostered, the wellbeing of children throughout the world will be remarkably improved."

Human Reproduction and Contraceptive Research

The exciting advances being made in reproductive biology will undoubtedly lead to safer, more effective family planning in both developed and developing countries, and a greater number of contraceptive options from which to choose. The relationship between heterosexual transmission of AIDS and choices in contraception must, however, also be considered.

With this introduction, Dr. Gary Hodgen, professor and scientific director at the Jones Institute for Reproductive Medicine of the Eastern Virginia Medical School, described the state of contraceptive technology today and the new research areas that will garner attention in the future. He also described the Contraceptive Research and Development Program (CONRAD) being undertaken at the Jones Institute for Reproductive Medicine (see box).

About 70 percent of the population of the developed countries uses some method of fertility control, reported Hodgen, ranging from the highly effective to the moderately effective. In the developing countries - using Nigeria as an example - 5 percent or less of the population practices family planning. Thus, there is much to be done in the developing world. In undertaking such work, cautioned Hodgen, it is important to realize that contraception is not just a tool for limiting the number of births. It is also a tool for managing the spacing of births, thereby allowing healthy children to be raised in a more nurturing environment.

U.S. investigators are now on the threshold of a major conceptual change in the development of new methods of birth control. This change is based on prevention of the interaction between the egg and sperm that achieves fertilization (local effects), rather than suppression of the reproductive function (systemic effects). The idea is to render the egg or oocyte incompetent for fertilization and development, while avoiding any effect on hormone production by the ovary or the testes. Achievement of a local effect avoids the host of side effects that result when hormone levels are altered - for example, problems with bloodclotting factors, liver metabolism, skin, and cancer, just to name a few. The sperm cell may be another selective site of action, particularly the site of the epididymis where sperm cells gain fertilizing capability, rather than the spermatogenic epithelium within the testes itself where hormone production might be affected.

Contraceptive Research and Development (CONRAD) Program

The Jones Institute for Reproductive Medicine at the Eastern Virginia Medical School is the primary center of the CONRAD program, which began in the fall of 1986 with funding of $28 million and the support of USAID. The objectives of the program are to develop and identify technologies useful in family planning and to provide the scientific basis for their safety and mechanisms of action.

The intramural research effort (30 percent) consists of the basic studies and phase one and two clinical trials, while the extramural research effort is undertaken off campus in conjunction with the Population Council, Rockefeller Foundation, Mellon Foundation, World Health Organization, and National Institutes of Health, among others. When possible, projects that may result in improved means of contraception are organized with investigators from the United States and, especially, Third World countries. Additionally, the assistance of pharmaceutical and biotechnology companies is sought in the development of new products.

Another contraceptive possibility is GnRH (gonadotropin-releasing hormone), a 10-amino decapeptide made in the hypothalamus, located at the base of the brain. In the use of GnRH analogs for contraception, complete suppression of hormone production is being avoided, Hodgen emphasized, because of all the side effects created. The male would lose libido, for example, while in a female the severe hypoestrogenic status that would result from daily treatment could promote the risk of osteoporosis in young women. Fortunately, the early data reveal that intermittent administration of the GnRH antagonist will halt the production of a fertilizable egg, while maintaining the hormonogenic function (estrogen production) of the first half of the ovarian menstrual cycle. Oral progesterone or some other progestin must be provided, however, to avoid an "unopposed estrogen status" and the risk of indomitable carcinoma. An orally active form of progesterone is now being tested because many of the progestins have undesirable side effects, such as androgenization. Some progestins do not have the desired selectivity or specificity, and they affect hair growth, cause acne and weight gain, and shift important HDL (good cholesterol) toward LDL (bad cholesterol). A new progestin, Norgestimate , has a low androgen effect and a selective progesterone-like action on the uterine endometrium. It is now in phase three clinical trials.

Hodgen also mentioned the research being undertaken on nonsteroidal gonadal hormones such as Inhibin, a heterodimer which can produce inhibition of follicle-stimulating hormone secretion from the pituitary.

Acquired Immune Deficiency Syndrome (AIDS)

The relationship between human sexuality and a high rate of AIDS transmission - perhaps more than 20 percent of the population in some areas - means that researchers can no longer develop or advocate contraceptive methods without taking into account their impact upon transmission of the deadly human immuno-deficiency virus.

Since little is known about the vector sources or routes of HIV transmission among heterosexual couples, reported Hodgen, it is also not known what impact the contraceptive methods used today have on transmission - whether they have no effect, a greater effect, or a protective effect. "It is imperative that we get those answers as rapidly as we can," declared Hodgen. "I think it is appalling that we have come as far as we have looking at this cloud called AIDS transmission and we have done as little as we have." He advocated rapid action in this area and appealed for information on how well the heterosexual transmission of AIDS in developing countries is being tracked.

Of interest to Hodgen is the spermicide Nonoxynol 9, which is known to have some protective effect as a viricide. Although it was not designed to serve that function, observed Hodgen, perhaps reagents can be developed that are contraceptive through their spermicidal action while at the same time acting as a potent and reliable viricide. They can then kill viruses before they can enter the body and cause contamination with HIV.

In a related area Hodgen advocated aggressive pursuance of the SAIDS (simian AIDS) model. SAIDS is a series of viruses that attacks macaque monkeys, including the rhesus. The familiar rhesus monkey, Hodgen believes, would be a good model for understanding the roots and vectors of AIDS and for testing what might be a potentially useful viricidal agent to accompany a method of contraception. Hodgen called, moreover, for quick steps to evaluate the commonly used methods of birth control to determine the primary risk factors in terms of roots and vectors. He warned, however, that the public's perception would be important in any pronouncement that a particular method of birth control is vulnerable to AIDS transmission. The data must be on hand to back up any such statements, he stressed.

Animal Biotechnology

Genetic Improvement of Farm Animals

According to Dr. Tilahum Yilma, professor of virology at the University of California at Davis, genetic improvements in farm animals over the next decade will largely result from the manipulation of gametes and embryos, the use of transgenic organisms, and the use of a new class of genetic markers.

The techniques of embryo manipulation were originally used to study the dynamics of early embryogenesis. Today, however, these techniques - much improved over time - are being applied commercially, and improved farm animals are obtained by gene transfer, cloning, and in vitro fertilization. Thus, it is now possible to produce animals with specific traits in one gestation period, rather than having to use the time-consuming classical genetic techniques. Yilma described some techniques that will be used more frequently in the future:
· Nuclear injection, in which cloned genes are injected directly into the nucleus of an embryo for integration into the host DNA, requires special techniques for the achievement of gene injection, gene integration, gene expression, gene regulation, and inheritance of the injected foreign genes. Because the success rate of this procedure is considerably higher than that of cytoplasmic injection, it will probably be used more commonly in farm animal production.
· Nuclear transplantation is used to produce parthenogenetic, gyno-genetic, and androgenetic embryos, and could possibly also be used to clone embryos. The latter has been demonstrated successfully in amphibians, but there has been little success in its application to cloning mammalian embryos.
· Embryo splitting has been used widely in livestock to generate identical offspring. The individual blastomeres of embryos can give rise to normal young.
· Chimeras, formed by aggregations of cells from different embryos, have been used to study the regulation of early embryo development.
· Injection of sperm from foreign species, using micromanipulation techniques, into oocytes to form normal pronuclei has been carried out, but Yilma was unaware of any reported births from such a technique.

A transgenic animal is one in which foreign DNA has been stably integrated into its germ line so that a foreign gene is transmitted as a normal inheritable trait. Using transgenics, a genetic trait determined by a single gene, such as the gene for growth hormone, can be introduced into an animal by microinjection of the cloned gene directly into the pronucleus of a fertilized egg, or by the use of retroviral and other viral vectors. Diseases associated with single genetic defects could possibly be treated by introducing a normal counterpart of the defective gene. Transgenic animals could also be engineered for use as animal models for those human hereditary diseases for which no natural animal models have been identified. Finally, explained Yilma, transgenic animals give information not only about random insertion and expression of a cloned gene, hut also about insertion of the gene in a specific tissue and its regulated expression, as determined by specific enhancers and promoters.

Quantitative traits - those that are not determined by a single gene product (polygenic in nature) - may in the future be identified by a new class of genetic markers (restriction fragment length polymorphisms), which are detected using cloned DNA sequences as probes.

Animal Health

The use of recombinant DNA methods to identify and diagnose animal diseases in the next decade will be particularly beneficial for Third World countries. According to Yilma, this technology will most likely contribute to the following research areas: common disease alleles, alleles arising from new mutations, genetic linkage analysis, forensic application of allele markers, neoplasia-acquired genetic alteration, infectious diseases, and detection of sequences.

The four kinds of animal vaccines used today depend on modern biotechnological techniques. These vaccines are: subunit vaccines, in which only the component of the agent that induces protective immunity in the animal is used as the vaccine; recombinant DNA-derived vaccines; synthetic peptide vaccines; and anti-idiotype vaccines.

Animal Nutrition

Genetic engineering of microorganisms in ruminant digestive systems allows the use of hard (high-lignin) fibers in animal feed. Lignin has long been theoretically regarded as indigestible and is the main factor limiting the use of some forages. The genetically engineered organisms, however, are able to convert hard fibers into products that can be used by the host animal for energy.

Marine Animals and the Marine Environment

Biotechnology will also have a bearing on the future of aquaculture, which is being carried out in many developing countries. Among its contributions, biotechnology will permit the identification and cloning of fish, mollusc, alga, and crustacean genes. In those countries bordering seas and undertaking commercial aquaculture, such a contribution will increase the production of food as well as that of important chemicals and drugs based on marine life.

In a related area, it is known that the petroleum-based pollution of the marine environment has dire environmental and economic consequences. Engineering of a bacteria able to degrade petroleum products will be welcomed by all countries.

Plant Biotechnology

The "green revolution" in the United States and elsewhere has been fueled by three technological advances: (1) plant breeding, particularly wheat and rice; (2) breakthroughs in the use of crop chemicals for controlling weeds, for reducing losses during the growing period, and for improving grain storage; and (3) farm mechanization and the ability to till land faster and more efficiently. As a result of the careful selection and application of these technologies, many Third World countries have become self-sufficient in supplying their own food. Nevertheless, even given this enormous progress, the most important advance in agricultural production in this century is just emerging, supported by advances in agricultural biotechnology and information science (see the chapter on "Information Sciences and Communications Technology"). Dr. Robert Fraley, director of plant science technology at the Monsanto Company, focused on agricultural biotechnology in his symposium presentation, and more specifically on recombinant DNA and its use in the genetic modification of plants.

How Agricultural Biotechnology, Works

Technology for introducing genes into plants revolves around the use of the soil bacterium Agribacterium tumafaciens. This bacterium is able to bind to a plant cell and introduce part of its genetic material into the chromosomal parts. Once introduced into the chromosome, the DNA is replicated, and it eventually becomes part of the plant's genetic information. It is then passed on through the seed. With the help of research, this system--which was established in the early 1980s - has been modified and improved so that it can be used routinely. The refinements over the last three to four years have been "spectacular and explosive," according to Fraley.

One of the key aspects of this technology is its minimal use of laboratory resources and equipment. In a simple procedure, small pieces of leaf or stem are sterilized and exposed to the agribacteria in a solution. The bacteria then bind to the plant cells and inject the genes into the plant material. Subsequently, the plant material is cultured in the laboratory under defined and sterile culture conditions.

This technology is now being applied to over two dozen plant species and the list is growing. Almost all of the vegetable crops, a growing number of the oilseed crops, and some of the important legumes can now be routinely manipulated using this technology. The cereals, including wheat and rice, are not yet on this list, because the Agribacterium system used to introduce genes is not compatible with gene introduction into the cereals. There have been remarkable breakthroughs, however, in the technology for the introduction of DNA into the cells of corn, rice, and wheat, and reports of these are just starting to appear in the scientific press. Fraley predicted that within the next three to five years every major agronomic crop will be manipulable using gene transfer technology.

State of Agricultural Research

In describing the present state of agricultural research in the United States, Fraley noted the concentration of research in terms of funding and expertise in universities and the private sector. The emphasis is on improving production efficiency, not necessarily yield, as in the past. The critical factor now is the unit cost of production, reported Fraley. Differentiating commodity products and an increased market orientation using taste, texture, flavor, and nutritional value, for example, are also important. Finally, the environmental contamination and pollution that stem from some of the agricultural technologies are of concern to many.

U.S. efforts in agricultural research are compatible with the long-term objectives of research aimed specifically at Third World needs. For example, despite the continuous improvement in crop production through advances in plant breeding, plant pests, particularly caterpillars and beetles, cause enormous damage in tropical agricultural areas. Molecular biology, however, allows scientists to clone the gene that codes for an insect control protein and use gent transfer technology to insert the gene into Agribacterium, where it is introduced into plants. The plants then regenerate to form transgenic plants that produce this insect control protein in their leaves. In the United States, this biotechnology-generated protection against insects is considered a value-added trait that will replace a certain amount of chemical insecticide usage, and it is an opportunity for developing countries, as well.

Another example of research oriented toward the U.S. market but of benefit to developing countries is weed control, probably the single biggest factor supporting yield increases in U.S. crops. Researchers developing new weed control chemicals are increasingly sensitive to how these chemicals will affect the environment, and research in molecular biology is focusing on understanding how these chemicals work, improving them, and designing safer, more effective molecules.

Yet another research topic of particular relevance to Third World countries is plant disease, and especially plant viruses. Viruses cause huge crop losses, particularly in tropical areas. One recently developed technology is a kind of plant immunization strategy based on molecular biology. For example, by cloning just the coat protein gene for tomato mosaic virus (a rod-shaped virus which has an RNA nucleic acid surrounded by a coat protein) and engineering it into a plant, the steady state production of the coat protein somehow interferes with the replication when that plant is infected with the virus. Although the molecular mechanism is not known precisely at this point, emphasized Fraley, the resistance created is dramatic and is applicable to Third World agriculture.

Transfer of Biotechnology

In looking at the transfer of this technology, Fraley referred to a 1982 report by the Office of Technology Assessment which dealt with the impact of technology on agricultural production. It concluded that the biotechnology-based plant improvements he had briefly described will have a dramatic effect on Third World agriculture - greater, in fact, than on U.S. agriculture. The report predicts, however, a three- to five-year delay in the transfer of this technology.

Another factor supporting the accessibility of the newest agricultural technologies based on molecular biology is that these technologies are being provided to farmers in a familiar form - seed. "More than anything else, that is the reason why this technology will be accepted much faster than anyone predicts today," said Fraley. He also predicted that the same agricultural chemical companies and multinational seed companies now selling seed to 150 countries worldwide will supply farmers in developing countries with these value added packages of genetically engineered seed. "It will follow very much the same trend - in a different way, of course - that has developed in this country where private industry has taken over much of the responsibility of the agricultural extension system because it is a profitable, target-driven business."

In concluding, Fraley voiced concern that the impact of the advances being made in agriculture will not be fully exploited, largely because of the growing ambivalence in the United States toward agricultural research. The concern in the United States and around the world about the health and safety of agricultural technologies has resulted, Fraley believes, "in a general antitechnology movement that has caused confusion and has led to a poor understanding of risk-benefit issues in this case." He continued: "We are without a doubt on the verge of the most important agricultural advance of this century. It is really up to us how this technology will be developed' managed, and exploited."