Nonhuman Primates in Genetic Research on Common Diseases
The leading causes of death in the United States are heart disease and cancer, with diabetes ranking sixth (Minino and Smith 2001). In addition to the health burden of these multifactorial diseases, progressive chronic conditions such as osteoporosis are associated with significant costs for health care and quality of life (Siris and others 2001; Tosteson and others 2001). From a global perspective, parasitic diseases such as schistosomiasis and Chagas’ disease (American trypanosomiasis) present tremendous health burdens in developing countries (Chan 1997; Murray and Lopez 1997).
Genetic approaches to these leading causes of morbidity and mortality seek to characterize the genetic components that influence susceptibility to disease processes. Statistical and molecular genetic techniques are used to quantify genetic influences on disease-associated traits and, ultimately, to identify the specific loci determining patterns of variation. Knowledge of the genes responsible for susceptibility can be used to target treatments or recommend lifestyle changes (e.g., dietary restrictions) to the individuals most likely to develop disease. This information can
also facilitate drug discovery through identification of biological mechanisms to serve as novel targets in pharmacological development (Dykes 1996; Gelbert and Gregg 1997).
The dramatic progress in genetics that has occurred in the last decade has revolutionized the study of genetic susceptibility to complex diseases. The development of the human gene map, sequencing of the human genome, technological improvements that allow rapid large-scale genotyping of population samples, and advances in statistical genetic methods have created unprecedented opportunity for genetic research on common complex diseases.
For disease processes that occur at a frequency of 10% or greater, an analytical design utilizing extended pedigrees drawn at random from the population (i.e., not selected with respect to disease characteristics) is optimal (Almasy and Blangero 2000). The statistical power of this approach is a function of the size and complexity of the pedigree (Blangero and others 2000; Dyer and others 2001). Extended pedigrees that are not selected on the basis of disease phenotype are useful for analysis of any normal or disease-related trait that is common in the population. Thus, once genotype data are generated for a study of a given common disease, the pedigree becomes an invaluable resource for studies of other traits.
Nonhuman primate colonies frequently have complex pedigree structures, making them well suited to genetic analyses of common diseases. Nonhuman primates serve as excellent models for human disease studies because of their phylogenetic proximity to humans, the large degree of conservation of gene maps between human and nonhuman primates, the genetic and physiological similarities between humans and nonhuman primates, and the natural occurrence of many of the complex diseases that represent the greatest health burdens to the human population (VandeBerg and Williams-Blangero 1996, 1997).
Many complex diseases, including heart disease, diabetes, hypertension, osteoporosis, schistosomiasis, and Chagas’ disease, occur naturally in at-risk nonhuman primates. However, genetic studies in nonhuman primates should be directed toward those diseases for which nonhuman primates offer scientific advantages, rather than simply toward those for which nonhuman primates are suitable animal models.
For example, the baboon is an excellent animal model for studies of pathology and immunology in schistosomiasis (Nyindo and Farah 1999). However, it is not an ideal model for studying the genetic determinants of susceptibility to infection with Schistosoma mansoni. First, complex extended human pedigrees are available in areas that experience high rates of disease prevalence (Bethony and others 2001; Marquet and others 1996), whereas it would be impractical logistically and financially to subject the
number of pedigreed nonhuman primates needed for a genetic epidemiological study to a challenge with S. mansoni.
In contrast, the baboon is an excellent model for genetic studies of susceptibility to another parasitic infection, American trypanosomiasis or T. cruzi infection. Chagas’ disease is the leading cause of heart disease in Latin America, and it can result in a short-term acute illness or a long-term chronic condition characterized by progressive cardiomyopathy or megaesophagus and megacolon. While large extended human pedigrees with high rates of infection are available for genetic study (Williams-Blangero and others 1997), parallel genetic studies in baboons can shed light on the genetic determinants of pathology and progression of other correlates of infection that are impractical to quantify over time in a longitudinal human study (Williams and others 2000). For example, regular tissue biopsies and radiographic assessments are possible with nonhuman primates to a degree not feasible for human populations living in the remote rural areas where the disease is prevalent.
Nonhuman primates are ideal models for genetic studies of complex disease processes that have significant environmental components. For example, the level of dietary control possible with pedigreed nonhuman primates allows explicit assessment of the interactions between genetic effects and dietary effects in determining physiological correlates of heart disease.
Genetic epidemiological studies of atherosclerosis and its correlates in the baboon model provided the first documentation of a genotype by diet interaction effect for serum cholesterol variation in a primate (MacCluer and others 1988). This was the first explicit evidence for a genetic basis to response to dietary saturated fats and cholesterol. A Program Project from the National Heart, Lung, and Blood Institute (P01 HL28972) has supported research on the genetics of cardiovascular disease risk factors in the pedigreed baboon colony at the Southwest Foundation for Biomedical Research for the last 20 years. The initial documentation of a genotype by diet interaction effect on cholesterol variation by MacCluer and colleagues (1988) has subsequently been refined, and numerous aspects of lipoprotein variation in response to diet have been investigated (e.g., Mahaney and others 1999a; Rainwater and others 1998, 1999). With the completion of a baboon framework gene map (Rogers and others 2000) and the genotyping of all animals in the pedigreed colony for approximately 325 markers spaced evenly across the genome, linkage analyses are being pursued to localize and ultimately to identify the individual genes involved (Cox and others 2002). The discovery of genetic effects and dietary interaction effects on cardiovascular disease risk factors was made possible by the ability to experimentally manipulate the diet in the
baboon model, a technique not possible in large-scale studies of human populations.
Osteoporosis is a major health problem in the United States. One of the primary risk factors for development of osteoporosis is low bone mineral density. Genetic studies of this disease in human populations are hampered by the need to assess bone mineral density in the large numbers of related individuals required for genetic epidemiological analysis and the immeasurable variability in lifetime diet and exercise patterns. The same baboon population studied for the cardiovascular disease studies was assessed for bone mineral density, taking advantage of the pedigree and genotypic data generated for the population. Measures of bone mass and bone mineral density traits exhibit moderate to high heritability in baboons, with between 40 and 67% of the variation attributable to genetic factors (Kammerer and others 1995). A preliminary genome screen for genes influencing bone mineral density traits and other correlates of osteoporosis has localized genes with significant genetic effects on chromosomes 6, 11, and 12 (Mahaney and others 1997, 1999b). These results suggest that the baboon model will be informative for fully characterizing the genetic components of susceptibility to osteoporosis. As is the case with heart disease, knowledge of the genes involved in determining susceptibility may eventually allow targeting of diet and exercise programs to those likely to develop disease and may ultimately lead to new pharmacological interventions.
The examples above illustrate the great utility of nonhuman primate models for characterizing the genetic components of complex diseases that are major health burdens throughout the developed and developing world. Linkage analyses localizing genes with significant effects on trait variability have been conducted for a broad range of disease traits in the single pedigreed baboon population, indicating the tremendous value of a pedigreed and genotyped colony for biomedical research. Already, genes influencing risk factors for cardiovascular disease, Chagas’ disease, osteoporosis, hypertension (Kammerer and others 2001), and hormone levels (Martin and others 2001a,b) have already been localized in this population. Ongoing studies are assessing the genetic components of temperament traits (e.g., Kaplan and others 2001a,b) and correlates of psychiatric disease (Jeffrey Rogers, unpublished data) in the baboon model. Future research will focus on the detailed characterization and ultimate identification of quantitative trait loci.
Clearly nonhuman primate colonies have tremendous potential for use in the identification of genes that influence common diseases. However, the utility of nonhuman primate colonies for genetic research relies on several key sets of data. First and foremost, detailed pedigree records must be available. If single male breeding groups are used, pedigree data
alone may be sufficient for reconstruction of pedigrees to be used in genetic epidemiological analysis. If multimale breeding groups are used, genetic marker data will be critical for resolving paternity and maternity errors.
The identification of specific genes with significant effects on disease traits requires linkage analysis of genetic marker data in conjunction with disease trait data and information about the distribution of genes across chromosomes. Although a gene map exists for baboons, none is available for any other nonhuman primate species. The development of gene maps for nonhuman primate species commonly used in biomedical research will be another critical step in developing genetic research with nonhuman primates.
Just as the human genome sequence has been extremely informative for investigators trying to move from gene localization to gene identification in human studies, genome sequence data will be extremely valuable for genetic research with nonhuman primates. The priority for sequencing should be placed on the species most commonly used in biomedical research and, particularly in genetic epidemiological research, the baboon and the rhesus macaque.
There is tremendous potential for future genetic research on nonhuman primates. However, progress in genetic research with nonhuman primates will require a significant investment in the development of pedigreed colonies of nonhuman primates, genotyping and gene mapping efforts in species to be used for genetic research, and the development of sequence information for genetically well-characterized species. Genetic management and improvements in genetic resources will be critical if the benefits of the genome revolution are to be fully realized in research with nonhuman primates.
Research reviewed in this article was supported by NIH grants P51 RR13986, P01 HL28972, and R01 RR08122.
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MacCluer, J.W., Kammerer, C.M., Blangero, J., Dyke, B., Mott, G.E., VandeBerg, J.L., McGill, H.C. 1988. Pedigree analysis of HDL cholesterol concentration in baboons on two diets. Am J Hum Genet 43:401-413.
Mahaney, M.C., Blangero, J., Rainwater, D.L., Mott, G.E., Comuzzie, A.G., MacCluer, J.W., VandeBerg, J.L. 1999a. Pleiotropy and genotype by diet interaction in a baboon model for atherosclerosis. A multivariate quantitative genetic analysis of HDL subfractions in two dietary environments. Arterioscler Thromb Vasc Biol 19:1134-1141.
Mahaney, M.C., Czerwinski, S.A., Rogers, J. 1999b. Possible quantitative trait loci for serum levels of human cartilage glycoprotein-39 (YKL40) and osteocalcin (OC) in pedigreed baboons map to human chromosomes 6 and 12. J Bone Min Res 14(Suppl 1):S142.
Mahaney, M.C., Morin, P., Rodríguez, L.A., Newman, D.E., Rogers, J. 1997. A quantitative trait locus on chromosome 11 may influence bone mineral density at several sites: Linkage analysis in pedigreed baboons. J Bone Min Res 12(Suppl 1):S118.
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Genetic Considerations in the Management of Captive Nonhuman Primates
Genetic management is an important component of the management of nonhuman primate colonies regardless of whether the animals will be used for genetic or nongenetic research (VandeBerg 1995; Williams-Blangero 1993). Genetic management techniques can be used to maintain the long-term viability of nonhuman primate colonies for continued production of healthy breeders. In addition, genetic management approaches can be used to generate well-characterized research subjects for genetic studies and to allow selection of optimal groups of unrelated animals for use in nongenetic experimental protocols.
Genetic considerations are important at multiple levels for the management of captive nonhuman primate populations. Genetic variability between subspecies, between geographic groups, and within populations has important implications for both management and research (Williams-Blangero and others 2002). The genetic characteristics of the breeding population significantly influence the productivity and stability of a captive nonhuman primate colony.
GENETIC VARIATIONS BETWEEN SUBSPECIES
Subspecies of nonhuman primates are not always easy to distinguish on the basis of physical characteristics. However, significant genetic differences between subspecies may affect the reproduction rates in mixed-subspecies populations and the experimental utility of hybrid animals (Kohn and others 2001; Moore and others 1990; VandeBerg and others 1990b; Williams-Blangero and others 1990). Genetic variation between subspecies as assessed by genetic markers is expected to be reflected in differences for biomedically relevant traits between subspecies.
For example, significant genetic differences exist among the baboon (Papio hamadryas s.l.) subspecies maintained at the Southwest Foundation for Biomedical Research (Williams-Blangero and others 1990). The genetic distances between the subspecies are mirrored by differences in phenotypic variability for lipoprotein traits (Williams-Blangero and Rainwater 1991; Williams-Blangero and others 1990). The baboon population at the Southwest Foundation is being used in ongoing genome scans for genes influencing risk factors associated with cardiovascular disease and osteoporosis (VandeBerg and Williams-Blangero 2002). The animals are predominantly olive baboons (P.h. anubis), but a significant amount of admixture with yellow baboons (P.h. cynocephalus) has occurred in the past. Subspecies admixture, as measured by percentage of genes derived from the P.h. cynocephalus subspecies, has been identified as an important covariate in genetic analyses of both lipoprotein levels and bone mineral density (Mahaney and others 1995, 1999a).
Ideally, nonhuman primate colonies should be composed of a single subspecies. However, if admixture has occurred in the past, breeding histories can be used to estimate individual admixture in terms of percentage of genes derived from the less predominant species. This measure can then be used as a covariate in genetic analyses of data from hybrid animals, and as a means for identifying hybrid individuals to be eliminated from the breeding population.
GENETIC VARIATIONS BETWEEN POPULATIONS WITH DIFFERENT GEOGRAPHIC ORIGINS
Significant genetic differences that have biomedical relevance may exist between populations of the same species derived from geographically distinct regions. Therefore, it is important to consider between-population genetic differences even when subspecies are not formally recognized. The differences between rhesus macaques (Macaca mulatta) derived from India and those of Chinese origin clearly demonstrate the relevance
of considering geographic origin in structuring breeding colonies of nonhuman primates for biomedical research.
The ban on importation of macaques from India has resulted in the evaluation of rhesus monkeys of Chinese origin for the many biomedical research programs that rely on macaques, including those related to AIDS research. Significant differences between Indian and Chinese macaques have been documented for morphological, behavioral, physiological, genetic, and immunological characteristics (Champoux and others 1997; Clarke and O’Neil 1999; Joag and others 1994; Marthas and others 2001; Viray and others 2001), suggesting that these two types of rhesus monkeys should not be interbred in captive colonies, except for special research purposes. Recent work by Marthas (Marthas and others 2003; Marthas and others 2001) has shown that colonies of Chinese-origin rhesus macaques may be of significant utility for AIDS-related research, despite differences from rhesus macaques in immune response to simian immunodeficiency virus.
GENETIC VARIATION WITHIN BREEDING POPULATIONS
The central goals of genetic management are to maintain genetic variability and to avoid inbreeding in order to maximize the long-term viability of captive populations. Achieving the goal of maintaining genetic variability obviously requires that colony managers be able to assess genetic variability. Genetic marker data for a large number of loci provide the most direct means of assessing genetic variability and can be used to estimate and monitor levels of heterozygosity in the population (e.g., Morin and others 1997). When genetic marker data are unavailable but the pedigree is known, genetic variability can be assessed from estimates of the genetic variance in phenotypic traits (such as clinical chemical and hematological traits), which are routinely included in animal colony records (Williams-Blangero and others 1993; 1994). Alternatively, information about the existing pedigree structure alone may be used to estimate genetic variability using computer simulation approaches (e.g., Caballero and Toro 2000; Dyke and others 1990; MacCluer and others 1986).
Genetic variability can be maintained by maximizing the effective population size, a process that minimizes the rate of loss of rare alleles (Kimura and Ohta 1969). The effective population size is essentially the breeding portion of the population, which can be increased by equalizing the genetic contributions of founder animals to the population. For example, the chimpanzee colony at the Southwest Foundation relied on a relatively small proportion of potential sires as breeders, resulting in a large variance in male reproduction (Williams-Blangero and others 1992). A computer simulation experiment demonstrated that if sires had been
randomly selected from the pool of available sires that were of breeding age and were unrelated to the dam, the effective population size of this colony would have almost doubled (Williams-Blangero and others 1992).
Inbreeding (mating between related individuals) can result in the loss of genetic variability and accumulation of deleterious recessive alleles. In nonhuman primates, inbreeding has been shown to increase morbidity and mortality and to decrease reproductive performance (Crawford and O’Rourke 1978; Noble and others 1990; Ralls and Ballou 1982). Colony managers should select unrelated mate pairs to avoid these effects of inbreeding depression. The cumulative effects of random inbreeding can be minimized by equalizing the sex ratio of breeders, maintaining genetic representation of founder animals, and bringing new unrelated animals into a colony (Williams-Blangero and others 2002).
PEDIGREE CONSTRUCTION, VERIFICATION, AND MANAGEMENT
The pedigree structure of a colony is the fundamental piece of information required for effective genetic management of the population, for selection of samples of unrelated experimental animals, and for genetic epidemiological research. Detailed colony records are essential for pedigree reconstruction. If single-male breeding groups are used, colony records may be sufficient to enable pedigree construction for genetic management purposes and for quantitative genetic analyses. Extended pedigrees can be reconstructed from colony record information on the individual’s identification number, the dam’s identification number, and the sire’s identification number for each colony animal (providing that each animal has a unique identification number) utilizing PEDSYS, a pedigree-based data management system (Dyke 1989).
However, it has long been recognized that errors in caging records and assignment of paternity and of maternity can occur even in the most carefully managed colonies (Curie-Cohen and others 1983; VandeBerg and others 1990a). Genetic marker information can be used to verify pedigrees constructed from colony records when paternity is thought to be known (e.g., VandeBerg and others 1990a). If multimale breeding groups are used, pedigrees can be reconstructed using genetic marker data provided that all of the potential sires for a given offspring can be identified and evaluated for mendelian consistency with the genotypes of the dam and the offspring. Pedigree reconstruction by exclusion of all but one potential sire for a dam-offspring pair has been successful in establishing pedigrees for multimale breeding groups of chimpanzees, rhesus monkeys, and vervets (Ely and others 1998; Newman and others 2002; Smith 1980; Vigilant and others 2001).
INCREASING THE VALUE OF PEDIGREED NONHUMAN PRIMATES FOR GENETIC RESEARCH
Pedigreed colonies of nonhuman primates are extremely valuable for genetic research. The statistical power of a genetic epidemiological study is a function of the size and complexity of the pedigrees included in the analyses (Almasy and Blangero 2000). Nonhuman primate colonies frequently have complex extended pedigrees that can be used in quantitative genetic analyses of variation in normal and disease-related phenotypes for both biomedical research and management purposes.
Generation of detailed genotypic information for extended nonhuman primate pedigrees as part of a genome scan is the ultimate way to increase their value for biomedical research. Provided that the families were not originally selected on the basis of a disease trait, a genome scan sample can be used for genetic investigations of any disease-related or normal trait that can be characterized in the population. A genome scan utilizes information on large numbers of markers spaced evenly throughout the genome in conjunction with a map of the markers to chromosomal locations and disease-related trait information, to localize the individual genes influencing the trait to specific chromosomal regions. The sample of baboons at the Southwest Foundation originally selected for a genome scan for traits related to osteoporosis has subsequently been used to localize and characterize genes influencing cardiovascular disease, hypertension, and levels of reproductive hormones (Kammerer and others 2001; Mahaney and others 1997, 1999b; Martin and others 2001a,b).
NONGENETIC INFORMATION REQUIRED TO IMPROVE THE VALUE OF PEDIGREED NONHUMAN PRIMATES
Detailed management and disease histories for pedigreed individuals can be invaluable for future genetic analyses. Management information, such as rearing history, may be critical in the evaluation of phenotypic traits. For example, breast feeding and formula feeding are known to have differential effects on thyroid hormone levels in infant baboons (Lewis and others 1993). Nursery rearing was a significant covariate in genetic analyses of lipoprotein variation in the Southwest Foundation’s pedigreed baboon colony (Mahaney and others 1993). Detailed animal histories enable the explicit evaluation of covariate effects, which may improve phenotypic characterization and consequently the power of genetic analysis.
Veterinary records of naturally occurring diseases may provide a rich resource for identifying new disease-related phenotypes for genetic analysis. The phenotypic data on risk factors for disease included in medical
records can facilitate preliminary analyses of genetic effects that can then be used to justify a full-scale genetic study of a given phenotype.
Genetic management is critical for the effective management of nonhuman primate colonies and for advances in nonhuman primate genetic research. To maximize the utility of nonhuman primate colonies for genetic research, it is imperative to maintain detailed pedigree information, clinical histories, and management records. The development of new gene maps for nonhuman primates will be essential for linkage analyses designed to localize disease genes in species other than baboons. Increased genotyping is needed to facilitate pedigree verification/reconstruction and future linkage analyses in existing captive nonhuman primate populations.
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Influence of MHC Gene Products on Immune Control of AIDS Virus Infection: Consideration for Use in Nonhuman-Primate Resources
Thomas C. Friedrich, BS, and David I. Watkins, PhD
The global HIV pandemic is now entering its third decade. As of late 2000, there were more than 35 million people infected with HIV worldwide, with almost 70% of infected individuals residing in Sub-Saharan Africa. Although there have been extraordinary recent advances in treatment of HIV infection, the pharmaceuticals that mediate these treatments remain available only to a small percentage of the world’s individuals infected with HIV. Indeed, the burden of infection is borne predominantly by developing nations in which access to antiviral drugs and indepth clinical care is negligible. Therefore, there remains an urgent need to develop an effective prophylactic vaccine against this virus.
Crucial insights about AIDS virus pathogenesis and vaccine efficacy have come from the simian immunodeficiency virus-infected rhesus macaque, the best available animal model for HIV infection of humans. There is every reason to believe that this model will continue to provide a basis for fundamental understanding of virus-host interactions in HIV. Recent results from our laboratory and others have shown that the major histocompatibility complex (MHC) class I genotype can have a dramatic effect on immune control of infection with highly pathogenic immunodeficiency viruses. Moreover, in recent years, several groups have made important contributions to our understanding of the properties of Mamu-
Wisconsin National Primate Research Center, Madison, Wisconsin
A*01, a common MHC class I molecule responsible for some macaques’ relative ability to control AIDS virus infection. However, as a result of the careful analysis of Mamu-A*01 and the cellular immune responses it restricts, there is now an acute shortage of macaques expressing this molecule available for further study. Efforts to understand the influence of immunogenetics on AIDS in the macaque model must therefore be broadened to include other common MHC class I and class II alleles. This understanding will not only increase the power of the macaque as a model for HIV disease but will also ensure that valuable animal resources are not depleted.
NATURAL HISTORY OF HIV/SIV INFECTION
Although the human and simian immunodeficiency viruses have been the subjects of intense experimental scrutiny since their discovery in the mid-1980s, little is known with certainty about the mechanisms of disease associated with these pathogens. In most individuals, the natural history of immunodeficiency virus infection is a triphasic process. Primary infection is characterized by a rapid burst of viral replication, with virus titers in both HIV-infected humans and SIV-infected macaques reaching higher than 107 RNA genome equivalents (copies) per milliliter plasma. Resolution of acute-phase viremia occurs within several weeks of infection, initiating a chronic phase, which can last from a few months to several years. This chronic phase is thought to represent a dynamic equilibrium, in which a balance is struck between ongoing viral replication and its partial control by the immune response, resulting in a steady-state or “set-point” in which virus burdens remain essentially stable. Finally, almost all infected individuals succumb to the infection, showing an increase in virus load and a precipitous loss of CD4+ T cells, resulting in an increased susceptibility to opportunistic infections.
ROLE OF CTLs IN THE IMMUNE RESPONSE TO HIV/SIV
The eventual death of most individuals infected with immunodeficiency viruses stands in apparent contradiction to the observation of strong humoral and cellular immune responses in most infected hosts. Recent advances in technology have greatly facilitated the study of the antiviral CTL response both in HIV-infected humans and in the SIV-infected macaque. Together, the many recent studies of CTL activity in immunodeficiency virus infection have suggested that CTLs play a major role in the modulation of the HIV- and SIV-associated disease course.
Because CTLs are apparently responsible for controlling virus replication in the acute phase, and because the value of the viral load at set
point strongly predicts the rate of disease progression, the strength of acute-phase CTL responses may “set the set-point” and largely determine disease outcome. Robust CTL responses present during the acute phase would then appear crucial, and indeed possibly sufficient, for control of infection. However, most infected individuals do progress to AIDS, despite vigorous CTL responses. One is then presented with another apparent contradiction: How can these viruses persist in a host in the face of a strong cellular immune response? Effective CTL responses likely exert selective pressure on these notoriously mutable pathogens. Could they vary their antigenic properties in such a way as to avoid immune detection? In fact, there is now overwhelming evidence that HIV, SIV, and other viruses are capable of spawning variants that allow them to “escape” from specific CTL responses, and furthermore that this escape can occur within the first weeks of infection.
IMMUNODOMINANCE OF SIV-SPECIFIC CTL AND CONTROL OF SIV INFECTION
Most studies of CTL activity against SIV in the rhesus macaque have centered on responses restricted by a single common MHC class I molecule, Mamu-A*01, expressed in ~20% of captive-bred macaques of Indian origin. For example, we characterized 14 epitopes derived from SIVmac239 bound by Mamu-A*01 recognized during chronic infection. Of these, CTL directed against the Gag CM9 epitope were present at the highest frequency, and were therefore considered immunodominant. In other studies, we determined that CTL specific for another epitope, Tat SL8, were present during acute infection at frequencies equal to those of Gag CM9-specific CTL. Tat SL8- and Gag CM9-specific CTL are thus equally immunodominant during the acute phase. In fact, CTL of these two specificities were the most frequent in all Mamu-A*01-positive macaques assayed, regardless of the other MHC class I molecules each animal expressed. Mamu-A*01-restricted CTL may thus reproducibly be dominant to other CTL responses in every Mamu-A*01-positive animal, irrespective of the MHC context in which Mamu-A*01 is expressed.
Immunodominance relationships are determined simply by enumerating the frequencies of antigen-specific CTL. Several recent technological innovations have simplified this task enormously; however, although it is now much less technically demanding to quantify antigen-specific CTL, the physiological importance of immunodominance relationships remains unclear. In the case of AIDS disease and pathogenesis, one might wish to determine which CTL responses are the most effective, that is, which responses are best able to eliminate virus-infected cells. These immune responses would make attractive candidates for targets of future vac
cines. The relationship between Tat SL8- and Gag CM9-specific CTL responses mentioned above may provide insights into ways of determining CTL efficacy, and it also may demonstrate the utility of the SIV-macaque model to the fields of AIDS pathogenesis and vaccine design.
“FUNCTIONAL AVIDITY” OF CTL AND SELECTION FOR ESCAPE MUTANTS
Faced with the observation that CTL specific for two Mamu-A*01-restricted epitopes appeared to be equally immunodominant during acute SIV infection, we sought to determine whether any differences exist in the impact of CTLs with these specificities on the virus population. As we had previously shown, CTLs that recognize the Tat SL8 epitope rapidly and reproducibly select for viral escape variants within 4 weeks of infection with SIVmac239. Conversely, although we and others have observed mutations within the Gag CM9 epitope that are consistent with CTL escape, these mutations appear in the viral population much later, beginning around 1 year after infection. Moreover, mutations in the Gag CM9 epitope do not appear to occur reproducibly in all Mamu-A*01-positive macaques. Because CTLs recognize these two epitopes exist in relatively equal frequencies during the acute phase of infection in most Mamu-A*01-positive animals, we reasoned that immunodominance alone cannot account for the differential ability of CTLs to eliminate susceptible viruses from the actively replicating population. Tat SL8-specific CTLs appear rapidly able to eliminate susceptible viruses, such that viruses with wild-type epitope sequences are not detected in Mamu-A*01-positive animals by 6 weeks after infection.
We hypothesize that this phenomenon can be understood in the following manner: Replication of the infecting (wild-type) virus population is reduced by an effective acute-phase CTL response. Rapid viral turnover then leads to a replacement of the wild-type sequence by mutant viruses, which have “escaped” this CTL response. The steady-state virus population measured during chronic infection would then represent mutants that have escaped the most effective CTL response. If this model is true, we would expect that the more rapidly the wild-type virus is eliminated, the more effectively the infection is controlled inasmuch as viral replication is contained more rapidly, allowing less time for the generation of escape mutants.
What mechanisms can account for the differential ability of CTLs to select for escape variants with rapid kinetics? Other investigators have shown that some CTLs require a lower density of MHC class I-peptide complexes on the surface of target cells to be sensitized to perform their effector functions. CTLs that are sensitized at relatively low peptide con
centrations were dubbed high “functional avidity” CTLs. It was proposed that these cells may be particularly effective at eliminating virus-infected targets because they could recognize these targets early in the infection process, when cell surface antigen densities were low.
We therefore determined whether CTLs detected in our SIVmac239-infected animals showed differences in “functional avidity.” We used titrations of epitope peptide (conc. 0.01 − 10000 nM) in interferon-gamma (IFN-γ) intracellular cytokine staining assays of fresh peripheral blood mononuclear cells to determine (1) the maximum IFN-γ output of CTLs recognizing different SIV-derived epitopes, and (2) the peptide concentration that sensitized these CTLs to release half the possible maximum of IFN-γ. CTLs with low half-maximum concentrations were therefore considered to be of high “functional avidity,” and CTLs requiring high peptide concentrations to reach half-maximum IFN-γ output were considered of low “functional avidity.” Strikingly, we found that Gag CM9-specific CTL required ~20 nM peptide, whereas Tat SL8-specific CTL required only ~0.1 nM peptide. We therefore conclude that Tat SL8-specific CTL demonstrate very high “functional avidity.” It is possible that this high sensitivity to their cognate epitope allows Tat SL8-specific CTLs to detect infected cells very early in the viral replication cycle that have only small densities of the Tat SL8 epitope on their surfaces, before the production of progeny virus particles. This explanation would help to account for the extremely rapid turnover of the viral population observed during acute infection in Mamu-A*01-positive animals.
SUMMARY AND RECOMMENDATIONS
The SIV-infected rhesus macaque remains the best available animal model for HIV infection of humans. Many important and incisive studies are feasible in macaques but would be impossible in humans. For example, our studies of viral evolution and the cellular immune response are predicated on our exact knowledge of the time, route, and dose of virus infecting the animals. Most importantly, we are able to challenge macaques with a pathogenic, molecularly cloned virus. This technique allows us to compare sequences of viral isolates obtained after infection with an inoculum that is both clonal and well defined, permitting us to dissect viral variation and evolution much more finely than is possible in human subjects. Knowledge of the infecting virus also allows us to design peptide reagents for cellular immune assays that reflect accurately the viral sequences present in infected animals, and limits the variation among experimental subjects in detection of these responses that is inevitable when using peptides derived from consensus HIV sequences on patient samples.
Moreover, fine analysis of selection on viral populations mediated by cellular immune responses also requires precise definition of viral epitopes, so that viral variation within and without epitope sequences can be accurately recognized. Unfortunately, this area of SIV research in macaques has, until recently, been relatively neglected. Complete data on peptide binding motifs are available for only a handful of macaque MHC class I molecules, and the SIV genome has been searched exhaustively for epitopes bound by only one molecule, Mamu-A*01, discussed above. Therefore, most studies of SIV-specific CTLs in macaques have hitherto focused on those responses restricted by Mamu-A*01, for which the most complete data were available. There is accordingly an acute shortage of Mamu-A*01-positive macaques available for research. We must use these valuable animal resources wisely and parsimoniously, to ensure their future availability.
One key way in which we can improve the macaque model of AIDS is to increase its similarity to the human clinical picture. To date, mucosal challenges with SIV have been carried out with large boluses of the virus so that all control macaques become infected after only one exposure. This system may overwhelm a potentially protective immune response induced by vaccination. In humans, it is thought that approximately 250 to 1000 unprotected sexual encounters with HIV-infected men are needed to cause HIV infection. There is thus a critical need to develop a low-dose intravaginal challenge model in the rhesus macaque. The shortage of female Indian macaques precludes this as a realistic goal for macaques bred in the primate center system. To develop a more physiologically relevant intravaginal challenge model, we will, therefore, import female macaques from China to initiate such studies in our primate center. We hope that these studies, and similar ones beginning at other primate centers, will help to develop and extend the utility of the rhesus macaque model for AIDS.
Indian- and Chinese-origin Rhesus Macaques for AIDS-related Research: Comparison of Vaginal Transmission Efficiency of Simian Immunodeficiency Virus (SIV), Viral Loads, and Virus-specific Antibody Responses
The use of rhesus macaques as a nonhuman primate model for human HIV infection and AIDS has resulted in an unprecedented demand that has far exceeded the supply of domestically bred animals; thus, researchers must use monkeys from other sources. Most domestically bred rhesus macaques are derived from animals imported from India. Because Indian macaques can no longer be exported, China has become one of the most reliable sources for rhesus macaques. However, it has been reported that the clinical course of SIV infection is slower and more variable in Chinese-origin monkeys compared with Indian origin monkeys (Joag and others 1994). We designed a study to determine whether Chinese-origin rhesus monkeys are more resistant to infection after intravaginal (IVAG) SIV inoculation compared with Indian-origin rhesus macaques (Marthas and others 2001). The findings of this recently published study are summarized below.
RESULTS AND DISCUSSION
We found no significant difference in the number of animals infected after one or two IVAG inoculations for Indian-origin compared with Chinese-origin macaques. Thus, rhesus monkeys originating from both countries are useful for studies requiring SIV transmission and infection. However, consistent with results of our previous studies, two IVAG doses of SIV resulted in significantly more SIV-infected macaques than one IVAG inoculation (Miller and others 1990, 1992).
We also compared the level of viremia in SIV infection in Chinese-and Indian-origin rhesus monkeys during the first few weeks of infection. As previously reported, SIV RNA levels in plasma among SIV-infected macaques were variable, and the variation was greater among the Indian-origin than among the Chinese-origin rhesus monkeys. SIV RNA levels at 2 weeks postinfection (PI) in plasma of Chinese- and Indian-origin animals were found to be high but not significantly different. However, by 6 weeks PI, the plasma SIV RNA levels were significantly lower in Chinese-compared with Indian-origin rhesus macaques, despite large overlap in the range of viral loads among Indian- and Chinese-origin animals. Our result is consistent with earlier observations from smaller numbers of Chinese and Indian rhesus macaques inoculated parenterally with SIV (Joag and others 1994).
Anti-SIV plasma antibody levels were also more variable in the Indian-origin rhesus macaques; however, at 6 to 8 weeks PI, there were no significant differences in SIV-antibody titers for Chinese- and Indian-origin rhesus macaques. It is well documented that Indian-origin rhesus monkeys that fail to make an antibody response to SIV or SHIV infection have a rapid disease course (Daniel and others 1987; Kimata and others 1999; Lewis and others 1994; Lu and others 1998). Our study found that rapid progression to AIDS (i.e., within 3 months PI) occurs at similar frequency in SIV-infected rhesus monkeys of Chinese-origin (1 of 10) and Indian-origin (1 of 16).
We used a panel of 13 highly polymorphic microsatellite markers to assess the degree of genetic similarity between monkeys of Chinese and Indian origin. Consistent with expectations for geographically separate populations of a single polymorphic species, we detected the majority of alleles for the 13 microsatellite loci in both Indian-origin and Chinese-origin animals; however, some allele frequencies differed among Indian-and Chinese-origin animals as reported previously (Morin and others 1997). We found no microsatellite alleles that were diagnostic for Chinese or Indian origin.
Overall, we found that the geographic origin of rhesus macaques does not predict the efficiency of vaginal SIV transmission or the level of
SIV RNA in plasma of SIV-infected animals during the first few weeks after IVAG inoculation. Most importantly, our results demonstrate that both Chinese-origin and Indian-origin rhesus macaques are well suited for AIDS-related studies that require mucosal SIV infection.
This work was supported by Public Health Service grant RR00169 from the National Center for Research Resources; grants AI39109 (M.L.M), AI39435 (C.J.M), and AI35545 (C.J.M) from the National Institute of Allergy and Infectious Diseases; and Elizabeth Glaser Scientist award 8-97 (M.L.M) from the Elizabeth Glaser Pediatric AIDS Foundation.
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John L. VandeBerg—Session Chair, Southwest Foundation for Biomedical Research, USA
Sarah Williams-Blangero—Southwest Foundation for Biomedical Research, USA
Thomas Friedrich—Wisconsin National Primate Research Center, USA
Marta L. Marthas—California National Primate Research Center, USA
QUESTIONS AND ANSWERS
PARTICIPANT A: Dr. Friedrich, you referred to the similarity in the Mamu system to that in the human in terms of regulation of escape. Could you please give us a little more detail.
DR. FRIEDRICH (Thomas Friedrich, Wisconsin National Primate Research Center): I will give you as much detail as I can. I think Dr. Marthas might like to comment as she alluded to some of the similarities between the human MHC system and the rhesus in her talk.
Basically, at a very simplistic level, there is a high degree of similarity. We do see selection for escape variant viruses by certain specific CTL in the human system. The escape is much more difficult to define in humans because they are infected with a heterogeneous population of viruses. By the time you get a human subject to study, it is usually too late to be able to define the actual genotype of the infecting strain of virus. It is much
more difficult to compare the viral sequences that you isolate from humans with earlier strains and to define this kind of thing.
As opposed to the macaque system, we can knowingly infect them with clonal viruses. That said, as Dr. Marthas discussed, there are certain alleles in the human that are associated with either a susceptibility to a rapid progression in AIDS or a resistance. HLA B-27 and B-57, for example, are associated with slow progression.
There are reports of human long-term nonprogressors, as they call them, which look clinically like the three SIV controllers that I described. They have low virus loads in the chronic phase of infection. They often have antigen-specific CD4 responses, which you would not normally detect. They have CTL responses that you might expect would have selected for escape variance, which they do not seem to have done, and so on. At that level, there is definitely a high degree of similarity between our models of what goes on in HIV-infected humans.
DR. KRAISELBURD (Edmundo Kraiselburd, Department of Microbiology Medical Sciences, Puerto Rico): Congratulations to individuals on the Genetics Panel for an excellent presentation. My question for Dr. Marthas is, do you have any data in terms of the disease preparation in the Chinese (rhesus)? In other words, we know there is a relationship between the nadir; is there progression? I have seen some data for the rhesus macaque, which is a slow progressor.
Second, of the rhesus that we have in Puerto Rico, 20% are Mamu-A*01 positive. I understand that some of your monkeys came from Puerto Rico. I would like you to comment on the fact that we did not see any relationship of the few monkeys that we tested in terms of disease preparation and Mamu-A*01 marker.
DR. MARTHAS (Martha L. Marthas, California National Primate Research Center): For this study, we did not measure long-term progression, which we originally began to titrate, so we purposely ended the study by 6 weeks. However, from other studies, the animals that have the controlled viremia, like the ones Dr. Friedrich described, are expected to continue long term. They have a more prolonged or delayed AIDS. If the animals, for whatever reason, had a high set point or did not control, they would have a more rapid progression to disease.
Dr. Mark’s group, which studies Indian versus Chinese, is similar although there are a couple of differences. First, they did an intravenous inoculation, which can make a difference in how the virus affects the animals. Second, they had a small sample size of Indians. If I had picked any two of my animals, I might have obtained a result similar to theirs. Their Chinese results looked very similar to ours initially. I would predict that if they were followed, some of those animals would have been very controlled.
The second question was A1 and why different groups see differences in the way Mamu-A*01 progresses. My hypothesis is that Mamu-A*01 may be a marker for something else. It happens to be convenient. I would predict that based on the human, there are linkages to equilibrium; however, there are associations that are nonrandom between Class 1 and other alleles on that same chromosome. So it might be that Mamu-A*01, from different geographic locations originally (we do not know where all of our Wisconsins or Indians came from) might be associated with something else that is controlling. Our Mamu-A*01 is tracking from Mamu-A*01, but it is not associated with whatever the other important alleles are tracking.
DR. FRIEDRICH: I agree with Dr. Marthas. I told you about our second experiment: we inoculated another set of three Mamu-A*01/ Mamu-B*17 double-positive animals. Because our laboratory does a lot of work on CTL responses, we were fervently hoping to find recapitulation of the scenario that took place with our first set of Mamu-A*01/Mamu-B*17 animals. Unfortunately, the animals did not control.
As Dr. Marthas was saying, although certain MHC Class 1 molecules basically may be markers for the ability of these animals to control, and we may be able to understand the ways in which these molecules may contribute to the overall control that we see, we simply do not know enough yet about macaque genetics to identify with certainty the other factors that might be influencing this ability. The experiment we performed recently shows that it is not only two MHC Class 1 alleles which is not surprising but is still disappointing.
DR. ROBERTS (Jeffrey Roberts, University of California at Davis): I have two questions pertaining to issues of population management. First, Dr. VandeBerg, in terms of the desire to maintain large populations of animals for pedigree, does the population at Southwest contribute to the availability of aged animals down the line, both in terms of genetic studies and availability of cost-effective production of aged animals? Second, Dr. VandeBerg or Dr. Williams-Blangero, in large multimale groups particularly at Southwest, are you concerned about having these large populations and about very cost-effective housing? If you have determined that there are males that have not contributed to the gene pool effectively for social reasons or whatever, do you advocate strategies to harvest those males at certain points either for indoor-timed mating or for other means of assisted reproductive technology to maximize their contribution?
DR. VANDEBERG (John VandeBerg, Southwest Foundation for Biomedical Research): With regard to aged animals, indeed, by maintaining these pedigreed animals throughout their lives, clearly we do end up with animals that have reached the end of their useful reproductive careers. We channel them into a specific group that we call the pedigree
geriatric baboon colony. We actually have more than 200 baboons in that colony now that are older than 17 or 18 years. Some are nearly 30 years old! We are developing some new research programs to study the aging process in those animals. They all are pedigreed and genotyped, and they will have been breeders to get to that age. They will have many progeny—grand progeny and perhaps great grand progeny—in the population. They are an extraordinarily valuable resource. It costs nothing to get them into the geriatric colony in the sense that they were productively used for research and breeding throughout their useful lives.
As for equalizing numbers of progeny from breeders, one of the most effective ways to deal with that situation is to harvest selectively. It may not be necessary to equalize the number of progeny of various males or females, but what you harvest for terminal experiments are an unequal number of progeny from particular parents so that you are very careful when you save back your breeders that you have equalized the genetic contributions from your females and from as many males as you can use in the particular breeding scheme. We effectively follow that process with our colony.
DR. WILLIAMS-BLANGERO (Sarah Williams-Blangero, Southwest Foundation for Biomedical Research): For the pedigreed section of the colony, we use all single male breeding.
DR. LYONS (Dr. Leslie A. Lyons, University of California at Davis): Dr. Friedrich, I think Dr. Marthas’ point about linkage to equilibrium is extremely valid. Do you know how your monkeys were related in either of your studies?
DR. FRIEDRICH: The monkeys we used for that study were not related to each other. Beyond that, I really cannot tell you. We can find that information in our colony records because they are kept in an animal and sire-dam triplicate, as described in previous talks. There is no easy way for us to go beyond that and determine what other genetic factors might be playing a role in these animals, short of sequencing their entire MHC side and finding out what happened.
DR. LYONS: Along those lines, would you suggest that while we are waiting for other sources of exports or identifying other populations of animals, we could genotype those animals and establish pedigrees so that we could bring in known different varieties of these animals to help our colonies. In other words, should we help those other countries get their animals genetically characterized?
DR. VANDEBERG: Such a plan would need to be carefully constructed with clear goals and a clear understanding of the actual potential for sending those particular animals to this country or to another country for research purposes.
DR. HEARN (John Hearn, Australian National University): First, Drs.
VandeBerg and Williams-Blangero, the issue of pedigreed and genotyped colonies is clearly a major added value that, spread more broadly, can not only give us high quality research and design but can perhaps also reduce the number of animals required in particular questions. Do you think we are at a stage where you could recommend a set of minimal criteria for genetic characterization and management of colonies in general as a separate issue to the kind of in-depth analysis that you would need for each specific disease, that has both cost and practicality connotations?
DR. WILLIAMS-BLANGERO: At a minimum, to begin constructing the pedigree only from the colony records gives you enough information to begin quantitative genetic analysis. With this information, which you can obtain from basic colony records for many nonhuman primate colonies, you can begin to ask the simple question: how much variation in this trait is attributable to genetics? As you ask these questions in conjunction with the existing phenotypic data on normal variation and traits in which you might be interested and on disease traits that are recorded in the clinical records, you can get an idea about productive directions for a true genome scan or more detailed genetic research. I think at whatever level you can feasibly do this, you enhance the value of the colony tremendously by adding any pedigree information. If you have genetic markers that are generated as part of other studies, and can contribute that information back into the colony to use for paternity testing and other purposes, it is of great value for enhancing the colony for genetic research.
DR. VANDEBERG: Let me add to that, however, that there are circumstances in which it is appropriate and cost-effective to produce animals in large breeding groups, such as our corrals or 6-acre breeding corral, which has about 600 baboons in it. I think the answer to your question must be tailored to the breeding situation, so there are situations where it is appropriate to have small breeding groups, large breeding groups, and so forth. Certainly, for genetic research, if it is economically feasible and practical to have single male breeding groups, that arrangement is by far the most valuable for genetic research, especially in the absence of markers to sort out paternity.
I think it would be difficult to give an overall set of minimum recommendations that would fit all breeding situations. I think they will have to be tailored to specific breeding situations and particular breeding objectives.
DR. HEARN: Thank you. My next question concerns emerging diseases—the next AIDS or, in particular, the transmission of viruses or zoonoses between nonhuman primate populations and humans. In captive colonies, or specifically in the wild where around the world there are particular areas where humans and nonhuman primates come into close
contact, and increasingly in areas where there is great ecological pressure, the situation is being set up for potential transmission. Are we ready, or should we be starting to investigate that issue?
DR. MARTHAS: I think it is important to set the boundaries and to let people know about this exposure. Participants at a recent meeting in Keystone, Colorado, were talking about HIV pathogenesis and starting to look at two different things: wild chimpanzees in preserves and SIV isolates. They are finding SIV isolates in chimpanzees using noninvasive methods like fecal and urine sampling. They are able to detect and indeed find these additional SIV isolates in populations that would not have been able to be tested before. By documenting and analysis, they are able to show that there are multiple crossover points from chimpanzees—multiple lineages of what is now an SIV that could potentially then go from chimpanzee to humans.
How did the SIV get into the chimpanzees? To make a long story short again, the answer is probably from other monkeys because chimps are known to hunt and eat other monkey species. That probability was, in fact, documented by a person looking and finding a variety of other species new SIV isolates in a variety of species that had not previously been found before. The more we look, the more we are finding it. There is more potential for human exposure and other nonhuman primates to retroviruses in this case, so we conjecture that it is exposure to any pathogen— parasites or other blood borne transmission.
With respect to your conservation question and human intervention, the most recent evidence is that the way the person sampled the 20 or more species of primates in Cameroon and central Africa was by going to bush meat markets. They sampled meat from monkeys that had been killed for human consumption. While consumption might not be the worst case scenario, certainly the preparing and hunting of those animals exposed the people and then exposed a broader population by eating the animals. I think it is very important, but we do not have an answer for how to deal with it.
DR. FRIEDRICH: Dr. Marthas lead into the current hypothesis for the transmission of HIV into humans. First, HIV began as zoonoses probably through the consumption of chimpanzees in bush meat that was infected with the precursor of HIV. We have this information from the work of Beatrice Hahn and Betty Korber. I do not know what primate centers and investigators of disease in nonhuman primates can do to be prepared for this kind of eventuality. We can only make public these findings so that people understand that by placing themselves and nonhuman primates at risk in these high-pressure ecological environments, it is more likely that we are going to find this sort of transmission occurring in the future. As Dr. Marthas said, the more you look, the more SIV
isolates you find, which is more potential for another zoonoses to occur in humans. In addition, there is every reason to believe there are other viruses we have yet to discover that could do the same thing.
DR. VANDEBERG: I would like to add that I think the primate centers and the primate community in general are actually very poorly prepared to deal with these issues in part because we do not have the facilities that are required. Our biosafety level 3 facilities are woefully inadequate in this country, and I am sure around the world, to deal with those kinds of issues. We do not have any biosafety level 4 laboratories capable of housing living animals. At our primate center, we are turning down studies that require biosafety level 3 facilities including studies on tuberculosis, anthrax, and West Nile virus. We are not meeting the current needs for lack of facilities, and we are certainly not in a position to meet the emerging needs that we do not even know of today.
The base grant budgets for the primate centers have been essentially flat over the 5-year doubling of the NIH budget. We have struggled to maintain what we have, not only in terms of facilities but also in terms of personnel. We are not in a position to recruit the personnel that are needed to establish the critical masses of scientists required for those kinds of investigations.
In regard to the potential of transmission of disease from chimpanzees, as we scale down the number of chimpanzees that are available for biomedical research, it is entirely possible that at some future point there will be another disease emerge, like AIDS or HIV. With the incredibly long generation time of chimpanzees, it would be very difficult to scale up that population if they were needed for research for a disease of that nature.
DR. ROBERTS: I would like to say that as we look at bringing additional primate sources into the United States, if we cannot genetically characterize them, one of the most crucial things is to establish the provenience of those animals. Where are they originating?
When you look at the range of facilities in China, if you can specify the geographic origin of those populations, it helps incredibly in terms of both looking at the genetics of those populations and also developing genetic tools to compare those populations.
DR. VANDEBERG: I would like to reinforce that point. The large variation that Dr. Marthas described in the Chinese rhesus may well be a consequence of geographic origin, a wide variety of geographic origin of animals. We talk about Chinese and Indian rhesus as if each population were homogeneous. Certainly they are not. Chinese rhesus from one part of China might behave very differently in these kinds of experiments than Chinese rhesus from another part of China. Thank you for mentioning that difference.
DR. ERWIN (Dr. Joseph M. Erwin, BIOQUAL and the Foundation for Comparative and Conservation Biology): I want to underscore the value of this morning’s presentations, particularly with regard to phenotypic characterization. The goals of genomics and the promise of proteomics cannot be appropriately realized unless there is a phenomic or phenotypic characterization component that parallels them.
We have heard that, fortunately, some of that characterization is going on. The support of partially characterized and pedigreed colonies is absolutely critical. Furthermore, I think there is the potential for some of the circumstances such as Mauritius, where there is a genetically homogeneous population that came from a relatively small number of founders and the St. Kitts African green population. Some of those island populations are essentially the best that we have in regard to inbred populations. We do not know what the potential is for genetic studies from those sources.
I think it helps to recognize that there is potential for working out some of the genetic risk factors and verifying them within the pedigreed colonies, then going to some of these relatively genetically well-defined biogeographic populations and selecting the animals that are appropriate for whatever the target research is. I think it could add some efficiencies, and one could even extend that selection to some of the other introduced populations such as the Macaca nigra population on Ba chang, the Macaca fascicularis population on Cabaña, the Macaca fascicularis population on Angour in Balow, and a number of other populations of this kind that could become tremendously valuable.
We must not neglect the other captive populations that exist but are currently not well supported. I think most of you are aware of a population of 300 chimpanzees that is now available and in need of support. I am trying to help develop such a support effort. So if you are concerned about this as much as I am, please contact me.
PARTICIPANT B: A message to take home is that making a switch to a whole different species would be of enormous consequence to a laboratory—even after establishing all of the norms and different information that we have on them, including the simple subtleties we see between the Chinese- and Indian-derived rhesus that sometimes are of great importance to a study. If you look at the other differences between the Chinese-and Indian-derived rhesus, you have differences in aggressiveness, serotonin, and alcohol consumption in blood chemistries. Those differences could be very relevant. Adding to what Dr. Erwin said, if you have a question on aggression, perhaps you would want to look at the more aggressive species as a good model. It really depends on what your model is in many cases if you are just beginning, but if you were already established, it would be quite difficult to make a switch.
DR. VANDEBERG: I agree it is very difficult to make a switch. I think it is not practical for an individual investigator to make that kind of a switch, particularly in the short term. However, if resources were committed to establish baseline data on some alternative species to rhesus over a period of time and those baseline data were developed and made available, it would be much easier for investigators beginning projects to choose a species other than rhesus. It may be possible for investigators at some point to switch, and it may be necessary if there are no rhesus available to them. However, the individual investigator is not in a position to make that switch by him- or herself. We must have support for developing the baseline data for many physiological characters for some of these other species that are readily available.
DR. PALMOUR (Dr. Roberta Palmour, McGill University): I would like to respond to Dr. Erwin about the monkeys on St. Kitts. First, we do have a pedigreed colony. We have been doing genetic studies within that pedigreed colony, which is quite large now. Second, we have done some work looking at the genetic variability on the island. Although it is certainly the case that the genetic variability is restricted by comparison with African vervets, there is a significant amount of genetic variability even from these 1000 founders. It is not that we have an inbred colony, but we do have a genetically restricted colony. I think one of the important points for the whole field is that by looking at different sources of variability, we will have models of different aspects of human traits. I think this point will be very important. I know Dr. VandeBerg and Dr. Williams-Blangero agree because they too have been doing this kind of work.
PARTICIPANT C: Is there a Macaca fascicularis equivalent to Mamu AO*1, and do you have any plans of expanding into cynos as an SIV model?
DR. FRIEDRICH: It depends on the equivalent. If you want to talk about an MHC allele that may or may not be associated with protection or relative control of SIV, it is quite possible. I am not familiar with any M. fascicularis data so I cannot tell you for certain. If you want to talk about an allele that would encode a molecule that would bind the same types of peptides or act in the same type of way, I would guess probably not.
DR. MARTHAS: I do not think there has been a systematic study, for instance, in M. fascicularis or other species. It could be done. I know from looking generally at a sequence database that there are similar sequences in MHC alleles where they have been studied for baboons, cynos, and rhesus. However, I do not think anyone has systematically gone through and tested them for whether you can obtain functional data or recognition by reagents. It is important to develop reagents that are either species specific or could work across multiple species.
DR. ERWIN: I also have asked several people that question because it
seems to me a very high priority that for African greens from St. Kitts and Mauritian cynos, this kind of work should be done. I appreciate that MHC is difficult to work with, but it seems to be a very high priority in the context of the discussions we are having at this meeting with regard to limited supply of rhesus.
DR. VANDEBERG: I would like to thank everyone in the audience for that wonderful discussion. It was extremely productive and exactly what we had hoped this session would become. Please join me in thanking the presenters.