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Animal models for AIDS by Mr. Pornpat Intarasunanont (Clinical group) Title: Animal model for AIDS. HIV clinical group of bioengineering and environmental health, An educational partnership between the CRI and MIT May 27 – July 24, 2000 By: Mr. Pornpat Intarasunanont Master degree student in toxicology program, Mahidol University. Out line Part 1 - Introduction Types of animal model Scientific purposes Selection of animal models The ideal animal model Part 2 - Classification of the models - Non human primate model - Immunobiology of human, chimpanzee, macaque monkeys - Study of lentivirus infection -Study of HIV-1 in chimpanzee -Study of HIV-2 in monkeys -Study of SIV -Study of non lentivirus model -Simian retrovirus type D (SRV-D) - Murine models -Lentivirus models -Transgenic mice and transgenic techniques -SCID mice (hu-PBL-SCID model & SCID-hu model) -Non lentivirus model -Murine leukemia virus (MuLV) - Feline models -Lentivirus model -Feline immunodeficiency virus (FIV) -Non lentivirus model -Feline leukemia virus (FeLV-FAIDS) Part 3 - Conclusion -Advantages and disadvantages between different animal models -Role of nef gene -Benefits and problems of using animal model -Extrapolation -Future prospects Part 4 - References 1 Pornpat Intarasunanont Introduction The acquired immunodeficiency syndrome pandemic has initiated large-scale efforts to understand the mechanisms of HIV pathogenesis and to develop antiviral therapies and vaccines. However, as in most virus infections, HIV pathogenesis is poorly understood and drugs or vaccines are still beyond the horizon. In this situation, the characterization of animal immunodeficiency viruses and the development and increasing understanding of animal models for AIDS is crucial if we want to better understand and combat HIV infections. Types of animal model The majority of laboratory animal models are developed and used to study the cause, nature, and cure of human disorders. Usually it is distinguished in the literature between four groups of animal models: induced, spontaneous, negative, and orphan models. The two first categories are by far the most important. As the name implies, induced models are animals in which the condition to be investigated is experimentally induced, e.g., the induction of diabetes mellitus with alloxan or partial hepatectomy to study liver regeneration. The induced model is the only category that theoretically allows a free choice of species. Although one might be tempted to presume that extrapolation from a species is better the closer this species resembles humans, phylogenetical closeness (as fulfilled by primate models) is not a guarantee for validity of extrapolation, as the unsuccessful chimpanzee models in AIDS research have demonstrated. It is just as decisive that the pathology and outcome of an induced disease or disorder in the tested species resembles the respective pathology of the target species. Feline Immunodeficiency Virus infection in cats may, therefore, be a better model for human AIDS than HIV infection in simians. A special type of induced disease models that are recently gaining increased popularity are transgenic animal models. Transgenic animals carry artificially inserted, foreign DNA in their genome. More recently, targeted gene replacement or "knock-out" models have appeared on the scene. Targeted mutation can be generated in a selected cellular gene by inserting mutated copies of the gene into cells and allowing one copy to take the place of the original, healthy gene on a chromosome. Such altered cells are helping researchers to produce mice carrying specific genetic mutations. For practical reasons, the preferred species for transgenic models is mice, but other species are now receiving growing interest. The welfare of transgenic animals has to be monitored with extra care as they may develop hitherto unknown disorders or be unable to express signs of distress, conditions that would render their further use in the experiment unethical and interfere with extrapolation. Spontaneous animal models of human disease utilize naturally occurring genetical variants. Many hundreds of strains/stocks with such inherited diseases, modeling similar conditions in humans, have been characterized and conserved. A famous example of a natural mutant model is the nude mouse, which meant a turning point in the study of heterotransplanted tumors and, e.g., enabled the first description of natural killer cells. The advantages/options of the nude mouse as an athymic animal 2 Pornpat Intarasunanont model were relatively quickly realized by the research community, whereas the nude (athymic) rat was known for almost 20 years before it was allowed into the immunology laboratories. Almost the opposite of spontaneous and induced images are negative models in which a particular disease does not develop, e.g., gonococcal infection in rabbits. Negative models also include animals demonstrating a lack of reactivity to a particular stimulus. Their main application is studies on the mechanism of resistance to gain insight into its physiological basis. The fourth term used to categorize animal models is the orphan model. An orphan model disease simply describes a condition that occurs naturally in a nonhuman species but has not yet been described in humans, and which is "adopted" when a similar human disease is later identified. Examples are Marek's disease, Papillomatosis, and bovine spongiform encephalopathy (BSE), the so-called "mad cow disease". Scientific purposes Today animal models are used in virtually every field of biomedical research both in preclinical and clinical studies. The aim of using animal models is to reconcile biologic phenomena between species, i.e. to examine system existing in one species and extrapolate knowledge to another. Researchers continually identify or develop new animal models to evaluate pathogenic mechanism, diagnostic and therapeutic procedure, nutrition and metabolic disease, and the efficacy of novel drug development. A common classification of animal experiments is according to scientific purposes. 1. To search for new knowledge 2. To diagnose diseases 3. To test new therapeutic techniques and new medicines 4. To detect and analyze drugs, hormones, and other biological compounds 5. To produce and test vaccines, sera, and other biological compounds 6. To test for toxicity, carcinogenicity, and teratogenicity of new and old drugs and chemical compounds Selection of animal models It is virtually impossible to give specific rules for the choice of the best animal model, because the many considerations that have to be made before an experiment can take place differ with each research project and its objectives. Nevertheless some general rules can be given: appropriateness as an analog transferability of information genetic uniformity of organisms, where applicable background knowledge of biological properties cost and availability generalizability of the results ease of and adaptability to experimental manipulation ecological consequences 3 Pornpat Intarasunanont ethical implications housing availability size of animal numbers needed life span sex amount of data needed age of animal progeny needed The criteria for selection or rejection of particular animal models include also customary practice within a particular discipline, diseases or conditions that might complicate results, the existing body of knowledge of the problem under consideration, and special features of the animal. The ideal animal model An ideal animal model for HIV and AIDS would be one in which 1. The test virus would be HIV itself 2. The host would be a small, inexpensive, genetically and immunologically wellcharacterized animal (e.g. mouse) 3. The target receptor would be CD4 4. The tropism would be comparable with that observed in humans 5. Disease development and 6. Mode of virus transmission would resemble the situation observed in humans Such an ideal HIV animal model does not exist. HIV-1 has been known for some years to infect chimpanzees and gibbon apes. Indeed, under special conditions it is possible to infect rabbits and even mice. More recently, infection of pig-tailed macaques (Macaca nemestrina) has been reported. Furthermore, HIV-2 infects rhesus and cynomolgus macaques, mangabeys and baboons. However, AIDS-related diseases in animals have not been reported following HIV-1 or HIV-2 infections. None of the animal species that can be infected with HIV meet all or even most of the criteria for an ideal animal model. Nevertheless they may serve as very valuable models to address specific questions in HIV pathogenesis and prevention, for example to analyze immune response mechanisms and the efficacy of experimental vaccines preventing infection. Classification of the models Concerning the animal species and the viruses that cause AIDS or AIDS-like disease in animal, the classification of the models can be separated mainly into 2 types of those retrovirus genera which are lentivirus (same genus with HIV) and other non lentiviruses (different genus with HIV but cause immunodeficiency in animal, mainly oncoviruses). Moreover there are 2 types of animal model prevalence in the study that are nonhuman primate models and murine models. Furthermore there are advantages to studies immunodeficiency in the feline or cats model. Each model consists of many types of viral infection and/or different 4 Pornpat Intarasunanont study aspects. A classification of each model is shown in the part 2 of this outline.(the models for non lentiviruses infection as shown are only the example among several types of those viruses and animal species) Table 1 Animal immunodeficiency retroviruses Prototype retrovirus strains Retrovirus subfamily Host Frequent clinical abnormalities Mechanism of immunosuppression ALV,REV Oncovirus Chicken B cell lymphomas, Induction of T suppressor spleen necrosis cells; T cell lysis LP-BM5 Oncovirus Mouse MAIDS, lymphoT and B cell dysfunction Duplan proliferation and lysis FeLV Oncovirus Cat FAIDS, anaemia, T cell dysfunction and lymphoma lysis FIV Lentivirus Cat FAIDS, respiratory, T and B cell dysfunction enteric infections and lysis MVV Lentivirus Sheep Encephalitis, pneumonia, None demyelination CAEV Lentivirus Goat Arthritis, encephalitis None EIAV Lentivirus Horse Anaemia, wasting, Mild T and B cell lymphocytosis dysfunction BIV Lentivirus Cattle Lymphocytosis Mild T and B cell dysfunction SRV Oncovirus Macaques SAIDS, diarrhoea, T and B cell dysfunction fibromatosis and lysis SIV Lentivirus Macaques SAIDS, lymphoma T and B cell dysfunction and lysis Figure 1 Phylogenetic relationships of lentiviruses are compared using pol gene nucleotide sequences for establishing phylogenetic relationships. Groups of primate lentiviruses are shown: HIV-1, HIV-2, SIVs, non primate lentiviruses: VMV, CAEV, EIAV, BIV, FIV. The scale indicates percentage difference in nucleotide sequence in the pol gene.The branching order of the primate lentiviruses is controversial. 5 Pornpat Intarasunanont Table 2 Clinical manifestations of lentivirus infections in natural hosts Lentivirus Disease description Ovine visna, maedi, and progressive Pneumonia virus Progressive lethal pneumonia, chronic encephalomyelitis, spasticity, paralysis, lymphadenopathy, mastitis, generalized wasting, opportunistic infections Generalized wasting, chronic leukoencephalomyelitis, progressive arthritis, osteoporosis, paralysis Fever, persistent viremia, hemolytic anemia lympho proliferation, immune-complex glomerulonephritis, bone marrow depression, central nervous system lesions. Persistent lymphocytosis, lymphadenopathy central nervous system lesions, weakness, emaciation Immunodeficiency-like syndrome, generalized lymphadenopathy, leukopenia, fever, anemia, emaciation, opportunistic infections Immunodeficiency, neuropathologic changes, wasting, opportunistic infections Immunodeficiency, lymphadenopathy, opportunistic infections, encephalopathy, emaciation, Kaposi’s sacroma and other cancers Caprine arthritis encephalitis virus Equine infectious anemia virus Bovine immunodeficiency-like virus Feline immunodeficiency virus Simian immunodeficiency virus Human immunodeficiency virus Non human primate model - Immunobiology of human, chimpanzee, and macaque monkeys infected with the immunodeficiency viruses. (Human) Three different phases can be distinguished in HIV infections in man. These are the acute infection, the quite, retracted infection of varying duration and the expanding immunodeficiency state. During the acute infection influenza or mononucleosis-like symptoms may develop but as in any other acute virus infection the immune system succeeds in effectively suppressing the replication of virus and in clearing the majority of infected cells. However, a complete clearance of virus is a rarity, if it ever occurs. During the phase of acute infection the immune system appears to function unimpaired. The second phase is the one in which there are no symptoms of disease and no signs of major damage to the immune system as reflected 6 Pornpat Intarasunanont for example in a reduction in the number of circulating CD4 positive T cells. Still there is evidence that the full range of immune reaction cannot be mobilized.It has been demonstrated that a subset of CD4 positive T cells that recognizes and responds to soluble antigens is selectively deficient at this stage. A polyclonal stimulation of B cells is seen and the capacity of the infected individual to mobilize primary immune responses is reduced. Probably there is also a gradual impairment of cell-mediated immune responses already at this stage presaging the final collapse of the system. The third phase involves dramatic changes in the immune defense system. There is a progressive reduction of CD4 positive T helper cells to very low levels. Antibodies against the internal gag proteins diminish and are substituted for by reemerging gag antigen. The virus infection is allowed to again disseminate in the body. (Macaques) Following infection with a viral pathogen, a complex series of cellular interactions occur resulting in an immune response which should control the spread of the agent and induce resistance to further challenges. Viral antigens are expressed on the cell surface in association with major histocompatibility complex (MHC) gene products where they can be recognized by T cells bearing antigen specific receptors. The generation of a specific immune response requires processing of viral antigens presented in association with MHC class II determinants on the surface of antigen presenting cells (APC) to CD4+ helper T cells and the production of the cytokine interleukin-1 (IL-1) by the APC. This results in the activation of virus-specific CD4+ cells, as evidenced by their proliferation and production of lymphokines including IL2, which mediate activation and differentiation of many other cells involved in the immune response. The activation of virus-specific CD4+ cells results in the differentiation of CD4+ T cells into effectors mediating delayed-type hypersensitivity, CD8+ T cells into cytotoxic T lymphocytes (CTL) or suppressor T cells, and the activation of monocytes/macrophages. In contrast to class II MHC restriction requirements of CD4+ helper T cells, the generation of CD8+ CTL from precursors requires recognition of viral antigen in association with class I MHC determinants and appropriate CD4+ helper T cell soluble mediators. Infected target cell recognition and CTL effector function occur in the same context. Table 3 Response of rhesus monkey to inoculation of virulent virus 1. Acute disease with rapid death 2. Subacute disease with later death 3. Chronic disease with long time survival and minimal evidence of clinical disease 7 Pornpat Intarasunanont (Chimpanzee) Inoculation of HIV-1 resulted in infection only in the chimpanzee, however at present, 6.5 years post infection, only two chimpanzees receiving high doses of HIV have demonstrated a temporary lymphadenopathy. Concerning the efforts to induce disease with HIV-1 in chimpanzees through the use of co-factors. Neither HIV-1 infection of hepatitis B or hepatitis C carrier animals nor experimental infection with cytomegalovirus and HIV-1 resulted in disease development. In addition, neither the use of immunosuppression nor immunoenhancement resulted in disease (table 4). This lack of disease development in the chimpanzee, with the exeption of the two cases of lymphadenopathy mentioned above, could be due simply to a time factor; the recently described average incubation period is 9.8 years in humans and has not yet been reached in the chimpanzee experiments. However, even if chimpanzees should develop disease, they still would not be a good model. It would simply be too much time in an experimental system. Table 4 Experimental co-factors to induce disease in chimpanzees Co- factor Effect Disease HIV dose T4 NANB, HBV CMV HIV Prednisone ATG WBC ,subsets , mitogens Anti-Leu2a T8 HIV Factor VIII HIV Allogeneic lymphocytes HIV Temporary LAD - Table 5 Inoculation of primate with HIV or SIV HIV Infection Disease Human Chimpanzee Rhesus Baboon + + - + - SIV Infection Disease N/A + + 8 N/A + - Pornpat Intarasunanont -Study of lentivirus infection Study of HIV-1 in chimpanzee Chimpanzee (Pan troglodytes) is the ape that has genetic similarity to human (about 98.5%). Some strains of HIV-1 can infect chimpanzees, thereby establishing an animal model to evaluate vaccines and infection with HIV-1. However, infection with most HIV isolates has not been accompanied by development of disease. After incubation periods exceeding seven years, several chimpanzees have shown signs of immune suppression and AIDS. HIV-1 from the affected animal may have become adapted to chimpanzee cells and developed a higher pathogenic potential. In vaccine studies utilizing the HIV/chimpanzee model, native or recombinant HIV env proteins and virus vectors carrying HIV protein genes have induced both cellular and humoral immunity. In addition, studies of env product vaccine candidates as well as more complex HIV vaccine strategies with recombinant adenovirus vectors or HIV DNA have shown efficacy in preventing HIV infection in chimpanzees. Hyperimmune anti-HIV immunoglobulin (HIVIG) and several monoclonal antibodies to envelope protein also have been evaluated for safety and potential to prevent HIV infection in chimpanzees under experimental conditions. Chimpanzee with persistent but apparently attenuated HIV infection have been exposed to other HIV isolates from the same or different clades in an attempt to learn whether immune responses that were not capable of clearing the initial infection were, nonetheless, capable of combating a new strain. Thus, infected chimpanzees may provide a valuable resource for studying cross-reactive immune responses. NIH supports chimpanzee colonies and programs that evaluate vaccine candidates and products for passive immunity in this animal model. The importance of using more pathogenic-virus challenge stocks and routes of exposure that more closely approximate naturally occurring HIV strains has been appreciated, and new stocks are in development or being tested for strains found both in the United States and elsewhere. Study of HIV-2 in monkeys The origin of HIV-2 has been traced to the sooty mangabey monkey (Cercocebus atys). This virus shares about half of the structural gene products found in HIV-1. HIV-2, which so far has mostly remained in Africa, does not cause disease as quickly as HIV-1, nor it is as easily spread. In fact, researchers have found one strain of HIV-2 that is more closely related to sooty mangabey SIV than it is to other HIV-2 strains. Some species of old world monkeys, e.g. pigtail macaque (Macaca nemestrina) and baboons (Papio sp) have found to be susceptible with infection of some isolates of HIV-2. The study of potential retroviral infection was investigated in macaques infected with human immunodeficiency type 2 (HIV-2) isolate (either GB122 or CDC77618). The findings demonstrate a window period for susceptibility to dual infection and indicate that protection from retroviral infection may be achievable. To assess disease progression in baboons (Papio cynocephalus) that were infected with two HIV-2 isolates ( HIV-2UC2, HIV-2UC14) and followed for 2-7 years period observation found that many of baboons exhibit lymphadenopathy, acute phase CD4+ T cell decline, and other signs of HIV-related disease. 9 Pornpat Intarasunanont Human immunodeficiency virus-2 –specific pathology in lymphatic tissues included follicular lysis, vascular proliferation, and lymphoid depletion. Both neutralizing antibodies and a CD8+ T cells antiviral response were associate with resistance to disease. Disease progression and the development of acquire immunodeficiency syndrome in HIV-2 infected baboons have some similarities to human HIV infections. The pigtail macaque (Macaca nemestrina) which is susceptible to infection with selected stocks of HIV-1 and HIV-2, and baboons, which continue to be studied. HIV-2 stocks have been developed that induce AIDS-like disease in Macaca nemestrina and baboons. Study of SIV SIV (simian immunodeficiency virus) constitute a group of lentiviruses that are indigenous to a number of healthy African monkey species, for example African green monkeys, sooty mangabeys, Syke’s monkeys and possibly chimpanzees. It appears that these African non-human primates represent the natural hosts for their respective SIV strains, as they are infected in the wild and remain healthy, indicating evolutionary adaptation between the virus and its host. Serological studies employing a variety of SIV antigens showed that additional African monkey species possess antiSIV antibodies and may therefore harbour yet to be isolated SIV strains. In contrast to the situation in African monkeys, feral Asian macaques do not seem to be infected with SIV. Macaque monkeys apparently became inadvertently infected during captivity either by biting or by veterinarian’s needles. SIV strains have now been isolated from rhesus, pigtail and cynomolgus macaques; (Macaca mulatta, Macaca nemestrina and Macaca fascicularis respectively). These heterologous hosts all develop a disease that shows many parallels to human AIDS. The similarities include CD4+ lymphoid cell and macrophage tropism, CD4+ cell depletion, development of opportunistic infections and tumours (in particular B cell lymphomas, but never Kaposi’s sarcroma), neurological manifestations and, as in humans, a humoral and cellular immune response that in the long run is functionally inefficient. Development of disease is more rapid in the macaque monkeys than in humans, facilitating studies of viral genetics, mechanism of pathogenesis, drug interference, vaccination and immunotherapy. Depending on the SIV strain used, on the dose and route of inoculation and on the recipient host species, SAIDS may manifest itself within a period spanning a few weeks to approximately 2 years postinoculation (or not at all if non-pathogenic virus isolates or clones are inoculated). SIVagm appear to constitute the most benign group, as none of the isolates has yet induced SAIDS following cross-species transmission, except possibly in one pigtailed macaque. This is all the more surprising as the variability among SIVagm isolates exceeds the variability within any other group of primate lentiviruses. Thus SIVagm may represent the oldest primate lentivirus group. The genetic organization of SIV is comparable to HIV, possessing analogous structural and regulatory genes (gag, pol, env, tat, rev, nef, vif, vpr and either vpu or vpx) and transcriptional control sequences (like TAR, RRE, Spl and NF-kB sites). Isolates from macaques are over 80% related to SIVsmm and to HIV-2 but only about 50% related to HIV-1. It has been suggested that a progenitor of SIVsmm (coming from a healthy natural host) may recently have given rise to the genetically closely 10 Pornpat Intarasunanont related SIVmac and HIV-2 strains, representing an example of cross-species infection of new heterologous hosts associated with disease induction. SIVmnd, SIVagm and SIVsyk are less closely related to the SIVsmm/SIVmac/HIV-2 group and are about equidistant to HIV-1 and HIV-2. The recently isolated SIVcpz of chimpanzees is the closest known genetic relative of HIV-1, yet again too distant to share a recent common ancestral virus with HIV-1. Thus, the evolutionary source of HIV-1 remains an enigma. Figure 2 Genome organization of primate lentiviruses. The linear double-stranded proviral DNA forms of HIV-1 and HIV-2/SIVmac show similar patterns of genomic organization. Structural genes (gag, pol, and env) are heavily shaded. Accessory genes, including essential regulatory genes (tat and rev), and nonessential genes (nef, vif, vpu, vpx, and vpr), are lightly shaded. The 5’ and 3’ LTRs flanking viral genes are shown as open boxes. Vpu is found exclusively in HIV-1, whereas vpx is found only in HIV-2 and certain strains of SIV. Vpr is encoded by both HIV-1 and HIV-2/SIVmac. The molecular cloning of pathogenic SIV demonstrated that SIV is both necessary and sufficient to induced SAIDS. More importantly, the availability of pathogenic clones should help to identify the viral determinants of pathogenicity either by mutational or recombinational (into a non-pathogenic SIV background) approaches. Due to the close genetic relationship of SIVs and HIVs and of their corresponding hosts and the similarities in disease development and manifestation, SIV in macaques is probably at present the most valuable (yet still expensive) animal model for AIDS research. On the other hand, one has to keep in mind that it is still a model, and overinterpretation of corresponding results may lead to errors. For example, SIV (and HIV-2) do not exhibit the V3 loop hypervariability of the HIV-1 env gene with its multiple epitopes for humoral and cellular immune responses, cautioning against generalization of results obtained from SIV vaccination experiments. 11 Pornpat Intarasunanont Another example: growth of SIVmac in permanent human T cell lines for use as whole virus inactivated vaccine has already led to results that are confusing or at least difficult to interpret: under certain experimental conditions, protection could be achieved even with uninfected lymphocyte immunogens. Also, a possible contribution of the MHC-polymorphism to the development of human AIDS may be difficult to investigate in outbred monkeys which have not yet been typed for their MHCpolymorphism. Conversely, one major advantage of working with the SIV monkey model has hardly been exploited so far, namely to ask the question of why natural host species like African green monkeys, sooty mangabeys and mandrills remain healthy despite persistent SIV infection. Learning the reasons for their continued health may greatly facilitate understanding of the pathogenic mechanisms triggered by HIV in humans. Table 6 Simian immunodeficiency virus isolates Primate species Rhesus monkey (Macaca mulatta) Pig-tailed macaque (Macaca nemestrina) Stump-tailed macaque (Macaca artoides) Cynomolgus macaque (Macaca fascicularis) Sooty mangabey monkey (Cercocebus atys) African green monkey (Cercopithecus aethiops) Mandrill (Papio sphinx) Syke’s monkey (Cercopithecus mitis) Chimpanzee (Pan troglodytes) * In wild-caught animals. Isolates Natural infection* Immunodeficiency induction SIVmac - + SIVmne - + SIVstm - + SIVcyn - + SIVsmm + - SIVagm + - SIVmnd + - SIVsyk + - SIVcpz + - 12 Pornpat Intarasunanont -Study of non lentivirus model Simian retrovirus type D (SRV-D) The prototype strain of SRV-D is the Mason-Pfizer monkey virus (MPMV) originally isolated from a rhesus macaque spontaneous breast tumour. SRV-D strains are not lentiviruses but instead belong to the oncovirus subfamily, although their oncogenic potential appears to be limited to the induction of fibromatosis. Correspondingly, the genomic organization of SRV-D is simpler than seen in lentiviruses with their regulatory gene, as SRV-D contain only the four gene gag, protease, polymerase and envelope. SRV-D are indigenous in feral Asian macaques and have been isolated from various species of captive macaques. SRV-D strains can be subgrouped into five distinct neutralization serotypes with differing pathogenic potential. SRV-D, even as a molecular clone, can induce simian AIDS (SAIDS) in (juvenile) SIV-negative macaque monkeys after short latency periods of 0.5-3 years. Non-pathogenic clones have also been described, recommending this model for the investigation of viral genomic determinants of pathogenesis. In contrast to the situation encountered with defective MuLV and FeLV oncoviral variants including immunodeficiency, pathogenic defective variants of SRV-D have not been observed. SRV-D induced SAIDS is characterized by wasting, chronic diarrhoea, retoperitoneal and subcutaneous fibromatosis and bacterial and viral infections, complemented at the cellular level by reduced lymphocyte response to mitogens, by T and B lymphopenia and anaemia. Long-term healthy survivors of SRV-D infections have been identified. The underlying reasons for their persistent health despite intermittent viraemia (in some animals) are not really obvious but may be correlated with the production of high-titre neutralizing antibodies. Induction of such antibodies with a formalininactivated whole SRV-D vaccine or with a vaccinia-env hybrid virus completely protected macaques against infection with an otherwise lethal challenge with live virus. Active and passive vaccine protection against SRV-D have been considerably more successful than what could be achieved so far in lentivirus animal models, possibly reflecting the much slower and more limited variant development of SRV-D in vivo. The viral host range is broader, including macaque B and T lymphocytes, fibroblasts and various epithelial cells. Natural transmission occurs mainly via saliva. The SRV-D model is particularly helpful in studying immunodeficiency induced by a widely occurring oncovirus. As molecular pathogenic and nonpathogenic virus clones are available, mutants and chimeric viruses will be acceptable tools to identify genomic sequences coding for disease potential. The model has already been of great value in vaccine studies. Infected monkeys may also be employed for therapy trails. Compared with SIV and FIV, the SRV-D model is probably not as relevant to human AIDS, as SRV-D strains are not lentiviruses, do not utilize the CD4+ receptor, have a different host range, induce protective neutralizing antibodies and exhibit a limited potential for the development of virus variants. 13 Pornpat Intarasunanont Murine models -Lentivirus models The high cost of primate models and the limited host range of HIV pose a challenge to researches, and have spawned many creative approaches to developing small animal models for the study of HIV-induced pathogenesis. If mice could be modified to model HIV-induced pathogenesis, they would make the ideal small animal model , as they are inexpensive to purchase and maintain, and their immune systems are well characterized. This review focuses on some of the murine models constructed to serve as small animal models for HIV-induced pathogenesis, and will highlight some of the lessons learned from these models during the past year. Transgenic mice Due to the lack of appropriate receptors for HIV, murine cells are refractory to HIV entry. To circumvent this limitation, investigators have created transgenic mice that express either the appropriate cellular receptors for HIV or contain HIV genes or proviral DNA in their germline DNA. While progress has been made with both models, considerable challenges remain. Initial attempts to construct mice permissive for HIV entry used either human CD4, which did not result in viral entry and productive infection, due to the lack of a required co-receptor molecule. Recently, several chemokine and chemokine-like receptors have been shown to serve as HIV co-receptor molecules. Macrophage-tropic strains have been shown to use CCR5 as the predominate receptor. T-cell-tropic isolates use the receptor for stromal cellderived factor 1, CXCR4, almost exclusively. Thus, attempts to create transgenic mice permissive for HIV entry were renewed, combining either CCR5 or CXCR4 with human CD4. By placing human CD4 and CCR5 under control of the T-cellspecific lck promoter, Browning created transgenic mice that expressed human CD4 and CCR5 in their CD4 and CD8 T cells. Despite the ability of peripheral blood mononuclear cells and splenocytes from these mice to support low-level viral production when infected in vitro, productive infection of transgenic mice could not be demonstrated following in vivo infection. However, polymerase chain reaction analysis of spleen and lymph node cells from infected transgenic mice revealed HIV-1 gag sequences, indicating that these cells were indeed permissive for viral entry. Similar results were found in transgenic mice expressing human CD4 and CXCR4 under control of the murine CD4 cis gene elements. This allowed expression of the transgenes in CD4 T cells, as well as CD4+/CD8– and CD4+/CD8+ thymocytes. In vitro infection of thymocytes and splenocytes with a T-cell-tropic strain of HIV-1 that resulted in low levels of p24 antigen mice was not reported. While the block to HIV entry may have been overcome in these new transgenic mice, these studies further support earlier data that indicate additional blocks to viral replication exist in murine cells. Reduced viral production in murine cells has been attributed to decreased activity of HIV regulatory genes such as tat and rev. These blocks to viral replication must be overcome before the transgenic models can be fully utilized. Cyclin T is a species-specific cellular factor recently identified in human cells, which potentiates tat activity when 14 Pornpat Intarasunanont transfected into murine cells, making this an excellent candidate for potentially alleviating the block to viral replication in a transgenic mouse model. An alternative approach to overcoming these blocks may be to utilize rabbits as a trangenic model. Unlike murine cells, the main block to viral production in rabbit cells appears to be primarily at the level of viral entry. Several transgenic lines have been constructed to contain either the full HIV-1 genome or various HIV-1-derived genes,unlike previous HIV-1 transgenics with less restricted transgene expression. To accomplish this, sequences derived from the CXCR4-tropic strain, NL4-3, were placed behind the human CD4 promoter. When the murine CD4 enhancer was included on the transgene, two out of five of the founders died by 5 months of age, and no transgenic lines could be established due to the early death or severe disease that occurred in the first-generation transgenics. These CD4C/HIV mice expressed the transgene at high levels in the thymus, and at moderate levels in the spleen and lymph nodes. There was a corresponding loss of CD4-bearing cells and disruption of architecture in these organs. The transgene was not expressed in cells that were not expected to express CD4. In addition to early death and disruption of lymphoid tissues, these mice exhibited other pathologies similar to those demonstrated in HIV-1-infected individuals, including severe wasting, tubulointerstitial nephritis and lesions of the lungs. These mice did not appear to die of opportunistic infections; rather, in the opinion of the investigators, wasting was the most likely cause of death. As in HIVinfected individuals, pathology was closely related to viral RNA load. Transgenic mice constructed without the murine CD4 enhancer remained healthy, demonstrated normal CD4/CD8 ratios and had approximately 20- to 60-fold lower levels of transgene RNA in their thymuses and spleens than did CD4C/HIV mice. In a subsequent study, transgenic mice constructed to contain various HIV-1 genes demonstrated that observed pathogenic processes. While transgenic cannot yet model the processes of infection such as viral spread and the emergence of viral variants, this approach may allow close examination of the disease processes occurring in tissues, and may also be useful in exploring therapeutic strategies. Opportunities in Transgenic Technology Interest in immunodeficient animals has mushroomed in recent years with advances in molecular biology and genetics that allow researchers to manipulate the genome of mice to eliminate or add genes and even replace selected genes. Science can actually engineer laboratory mice to meet specific research needs and protocols. Creating new, genetically engineered animal research models involves two transgenic techniques-microinjection of cloned genes randomly inserted into the host DNA and gene targeting or homologous recombination between cloned DNA and one of the identical copies of the sequence normally present in the chromosome. The more complete names of these two transgenic techniques are: 15 Pornpat Intarasunanont classical pronuclear microinjection: introduction of foreign DNA into embryonic pronuclei resulting in random integration and expression and embryonic stem (ES) cell-mediated gene targeting: introduction of genetically modified ES cells into recipient embryos resulting in the ablation (knockout) or modification of a specific genetic expression. Transgenic animals are designed to exhibit either a gain of function (expression of a novel cell-surface receptor) or a loss of function (knockout of a cellular function). Classical pronuclear microinjection techniques have been used for 15 years to create mouse models, which express unique phenotypes. The major flaw in the pronuclear microinjection models has been the random nature of transgene integration locus and copy number. Expression patterns may vary significantly in a series of lines expressing identical transgenes. Modifiers of expression such as age, sex and health status further confound the process, increasing potential for variability. By using ES cell gene knockout technology, an investigator can produce an animal model in which expression (or the lack of expression) is highly predictable. A clone of cultured ES cells is selected in which a specific DNA sequence in the mouse genome has been modified (usually inactivated). Transformation of cultured cells with foreign DNA is relatively simple and most commonly is achieved using a procedure called electroporation. All transgenic models, whether targeted or untargeted, still may present unpredictable expression patterns due to incomplete knockout of the targeted gene, redundancy within the genome or unanticipated genetic interactions, such as down-regulation of other genes. Despite some unpredictability questions, transgenic knockout technology can produce research animals that are "custom designed" to meet the specific needs of an investigator's experimental protocol. Knockout technology, or homologous recombination, is also a valuable tool for determining functions of specific genes. The hu-PBL-SCID model Other approaches to humanizing the mouse have relied on transplantation of human cells and tissues that normally serve as targets for HIV infection. One of these models is the hu-PBL-SCID mouse, which is constructed by the transplantation of human peripheral blood lymphocytes (PBLs) or cord blood lymphocytes into the peritoneal cavity of severe combined immunodeficient (SCID) mice. Since these mice lack mature B and T cells, the human cells are not grossly rejected and can engraft. Various strains of HIV can be introduced into this model, resulting in CD4 cell depletion. This system has proven useful for assessing viral pathogenic properties and passive immune therapies. Using the hu-PBL-SCID model, scientists have begun to address the question as to why macrophage-tropic isolates predominate during the course of HIV infection. They found a correlation between co-receptor usage, plasma viremia and CD4 cell depletion. In brief, macrophage-tropic isolates were slower to kill CD4 cells, they could sustain higher plasma viral RNA levels than either T-celltropic or dual macrophage/T-cell-tropic viruses and they had found the most rapid depletion kinetics. The higher levels of plasma viremia and the lower levels of cytopathicity would therefore result in a greater contribution to the viral pool by macrophage-tropic 16 Pornpat Intarasunanont isolates than either T-cell-tropic or dual-tropic viruses. As memory cells express higher levels of CCR5 than native cells, the authors argue that activation of the immune response by the transmitted virus would favor macrophage-tropic isolates. The authors further show that the switch from a macrophage-tropic isolate with low cytopathic ability to a dual-tropic isolate with rapid depletion kinetics is due to a single amino-acid change in the V3 region of gp120. Given the high mutation rate of HIV, the authors maintain that dual-tropic isolates most likely arise frequently but are simply outcompeted by the macrophage-tropic isolates. The ability to easily manipulate the hu-PBL-SCID model will no doubt allow further exploration of this important question. Several features of this model make it useful for investigations studying the efficacy of passive antibody administration against HIV infection and the study of antibody escape mutants. Passively administered antibodies distribute and equilibrate with the mouse serum, and have a half-life of approximately 1-2 weeks. In recent studies using CD4 IgG2 and monoclonal antibodies Bat 123, b12 and 694/98, all demonstrated 100% protection from HIV infection if administered prior to HIV exposure at does obtainable in humans. These monoclonal antibodies all recognize epitopes on gp120. Bat123 binds the V region of the gp120V3 loop, IgG1b12 binds an epitope that overlaps the CD4 binding site of gp120, and 694/98-D recognizes a linear epitope in the conserved GRAF motif at the tip of the V3 loop, but the epitope may have conformational components. Only Bat123 and b12 were shown to be 100% effective if given several hours post-exposure, but only b12 showed in vivo neutralization of primary isolates. This is in contrast to HIV-1-immune globulin fractions, which show only partial protection against HIV-1 and no protection against primary isolates. The potent post-exposure effect of b12 was not observed in vitro, which is most likely explained by the recent finding that it is complement mediated and requires the Fc region of the antibody. This latter finding demonstrates that the hu-PBL-SCID mouse has clear advantages over an in vitro culture model. One of the most urgent needs in AIDS research is for an animal model suitable for the investigation of primary vaccine strategies. The hu-PBL-SCID model is limited by the inability to consistently elicit primary immune responses. Approximately 1 month following introduction of PBLs into SCID mice, the T-cells become anergic and are unable to respond to mitogens or to CD3 stimulation. The limited VB repertoire, restricted CDR3 size distribution and high expression of activation markers observed in human T cells recovered from hu-PBL-SCID mice suggest that cells undergo the extensive antigenic stimulation and repertoire selection characteristic of a xenoreactive response. The lack of both human antigen-presenting cells and a suitable microenvironment play a major role in limiting primary immune responses in this model, although some reports of this have been made. The transplanted skin successfully engrafted in the majority of animals and survived for up to 1 year. Dendritic cells were identified in the skin as a potential source of antigen-presenting cells, but these cells were lost 3-4 months post-transplantation. Thus, the authors confined their experiments to the first 5 weeks post-transplantation of PBL. Intradermal inoculation of a canary pox vector encoding the 17 Pornpat Intarasunanont gene for HIV-1 gp160 resulted in in vivo priming of HIV-specific T cells. Although it is not clear how the T cells homed to the skin, perivascular infiltration following introduction of the vector. When interleukin 2 was co-injected, the infiltration in upregulation of homing receptors. CD4 T cell lines established from the infiltrating T cells were found to be of the Th-1 subset, and were HLA class II restricted, antigen specific and cytotoxic. However, no cytotoxicity was observed in the only CD8 T-cell line established from immunized mice, suggesting either a lack of in vivo CD8 T-cell priming or a more trivial problem with the in vivo culture system. These modifications are an encouraging start in advancing the utility of the hu-PBL-SCID model for vaccine strategies. The SCID-hu model An additional model that relies on the transplantation of human tissues into SCID mice is the SCID-hu mouse model. This model is constructed by surgical implantation of human fetal thymus and liver under the kidney capsule of a SCID mouse. The thymus and liver form a conjoint organ Thy/Liv that histologically and phenotypically resembles a normal fetal thymus. This Thy/Liv implant directs thymopoiesis from hematopoietic precursor cells for up to 1 year. Thus, this is a dynamic system that, unlike in vitro cultures of peripheral blood mononuclear cells, contains cells at various stages of differentiation and maturation. Infection of the Thy/Liv implant with HIV can be achieved with both molecular clones and primary isolates. Infection results in CD4 cell depletion, which, depending on the strain and dosage of virus, can be quite severe, resulting in hypocellularity, loss of corticalmedulary junctions and thymic involution. Like the hu-PBL-SCID model, primary immune responses have not been documented in the SCID-hu model. In addition, implantation of lymph nodes into these mice results in loss of architecture and activation of cells, making this model unsuitable for the study of both immune responses to HIV and HIV-induced pathogenesis in a secondary lymphoid organ. However, the SCID-hu mouse has proven suitable for the modeling of HIV-induced pathogenesis, and the assessment of both drug and gene therapeutic strategies. The human thymus has been shown to be susceptible to infection by HIV, and to undergo CD4 T-cell depletion in HIV-infected children. The majority of thymocytes expresses the CD4 molecule, and also expresses high levels of CXCR4. The thymocyte subset with the greatest percentage of cells expressing this molecule and expressing the highest levels of CXCR4 is the immature CD4+/CD8+, CD3, CD5 population. Using an HIV-based, CXCR4-tropic reporter virus, this subset of thymocytes has been shown to be the first to express viral proteins and to demonstrate the greatest CD4 cell depletion in the Thy/Liv Implant. This suggests that the levels of both co-receptor and primary receptor molecules play a role in the initial targeting of cells by HIV. Consistent with previous data obtained both in vivo and in vitro, CD4-/CD8+ mature thymocytes also expressed the reporter gene following infection of Thy/Liv implants with NL-r-HSAS. Infection of this subset in vitro was shown to be due to infection of cells at the CD4+/CD8+ stage, followed by differentiation into CD8 single-positive mature thymocytes. Export of these infected CD8 thymocytes to the periphery of 18 Pornpat Intarasunanont HIV-infected CD8 cells in the periphery of HIV-infected individuals. CD4 downregulation may also contribute to the observed virus expression in CD4 negative cells. Previous studies have shown that HIV strains containing deletions in nef are attenuated for replicative and cytopathic potential in both hu-PBL-SCID and SCID-hu mice, similar to that shown for SIV in macaques. Two recent studies demonstrated that previously identified regions such as the two SH3-binding proline repeat domains and the two arginines at positions 105 and 106 of nef, which have been shown to interact with various cellular kinases, are dispensable for pathogenic potential in these chimeric models. Rather, two different regions of unknown function spanning amino acids 72-75 of nef for hu-PBL-SCID and amino acids 41-49 for SCID-hu mice appear to be important. Further studies are required to determine how these regions are involved in Nef-mediated pathogenic effects. With the advent of highly active antiretroviral therapy to control viral replication, perhaps the most pressing question today is whether treated HIV-infected individuals will be capable of reconstituting the peripheral T-cell compartment. As the thymus is the primary site of T-cell development, especially in children, understanding the long-term impact of HIV on this organ is critical. Administered anti-retroviral drugs to infected SCID-hu mice following total depletion of CD4 cells. Regeneration of CD4+/CD8+ thymocytes was observed in all treated implants, demonstrating that HIV has no lasting deleterious effect on the thymic microenvironment, and that at least in pediatric HIV-infected individuals, the thymus may well be able to reconstitute the peripheral T-cell compartment. However, the observed reconstitution in Thy/Liv implants was transient. Recent studies by Amado et al, using a pathogenic reporter virus, indicate that virus breakthrough during the course of therapy is associated with the secondary decline of CD4 thymocytes. The mechanism for this breakthrough may therefore be due to an overwhelming viral burden that the drugs were not able to fully overcome at the dosages used. It must also be remembered that it is not likely that specific immune reconstitution following highly active anti-retroviral therapy. HIV infected individuals often exhibit multiple hematopoietic abnormalities such as anemia, thrombocytopenia, granulocytopenia, lymphocytopenia, monocytopenia and neutropenia. However, the mechanisms responsible for these HIV-induced abnormalities are not clear. Due to the presence of multilineage progenitors in the SCID-hu mouse and a suitable environment for the development of T cells, this is the only in vivo model for examining the impact of HIV on multiple human hematopoietic cell lineages. Recent studies have investigated the effects of HIV on multilineage progenitor cells in this model, finding a severe reduction of myeloid and erythroid colony-forming units (CFU) when cells from infected implants were plated on methylcellulose. While reduction in CFU were observed with both CXCR4- and CCR5-tropic viruses, CXCR4-tropic viruses were more aggressive in inhibiting CFU and inducing thymocyte depletion, correlating with a better replicative ability in the implant. Deletion of individual accessory genes also had no effect on inhibition of CFU, beyond affecting their replicative ability. However, inhibition preceded thymocyte depletion, suggesting a fundamental difference in the effects of HIV on colony-forming progenitors versus cells of the lymphoid lineage. Alternatively, this result may suggest that inhibition of progenitor cells interrupts thymocyte 19 Pornpat Intarasunanont development. However, one primary isolate, PT8MO, elicited a severe reduction of CFU without causing thymocyte depletion during the 6 weeks of the study, suggesting a differential effect of HIV on the two cell types. Jenkins et al, reported a loss of some CD34 cells; however, no sign of CD34 infection could be demonstrated in any of these studies. The most direct support for lack of CD34 infection comes from a new study in which Koka et al. Infected implants with a CXCR4-tropic reporter virus. No evidence of reporter gene expression was found in CD34 cells from these implants, despite severe functional defects of these cells. Taken together, these results suggest that the disruption of hematopoietic function is due to two effects, loss of some CD34 progenitor cells and perturbation of the extracellular microenvironment, such that hematopoietic progenitor cells are rendered non-functional. The latter scenario is supported by the finding that cells from infected Thy/Liv implants demonstrating reduced CFU, had a transient renewal of colonyforming activity after treatment of the implant with anti-retroviral drugs. This suggested that at least some CD34 cells are retained and function can be rescued. Further studies are now needed to define precisely define the cellular and/or molecular mechanism(s) of HIV-induced hematopoietic inhibition. The presence of progenitor cells in these implants and the directed thymocyte development also make this model well suited for the study of gene therapeutic strategies. If required, CD34 cells can be added exogenously, biopsies can be performed to determine the efficiency of gene retention, and the implants can be challenged with virus to determine efficacy. While several studies have utilized this system to study or optimize vector transduction strategies, these are outside the scope of this review. However, one study utilized the SCID-hu mouse to model efficacy of anti-HIV gene therapy. Bonyhadi et al. Recently used the SCID-hu model to test the HIV-1 revtrans-domonant mutant protein, RevM10. Rev is required for the transport of singly spliced and unspliced HIV-1 mRNAs to the cytoplasm of infected cells, making it critical for the expression of structural proteins. CD34 cells derived from either human cord blood or from granulocyte SCID-hu - Primary Lymphoid organ - Predominantly T-lineage cells with thymic stroma - Thymocytes exist at different stages of maturation and differentiation - Allows differentiation of T-lineage precursors - Predominantly CXCR4-bearing cells with a minority of CCR5 cells - CXCR4-tropic viruses are more aggressive in this model Hu-PBL-SCID - Peripheral lymphoid cells - Contains human cells of macrophage, T and B-cell lineages - T cells are predominantly mature and activated - Human immunoglobulin is present - Both CXCR4- and CCR5-bearing cells are present - CCR5-tropic viruses are more aggressive in this model Table 7 Comparison of the two major chimeric mouse models for HIV disease, showing some of the physical characteristics that must be considered when designing experiments. 20 Pornpat Intarasunanont Colony-stimulating factor-mobilized peripheral blood, which were transduced with a vector expressing both a reporter gene and RevM10, were able to implant in Thy/Liv implants and differentiate into mature thymocytes, as determined by disparate HLAA2 phenotypes. The transgene encoded the reporter gene, Lyt2, and expression of the transgene was observed in immature CD4+/CD8+ thymocytes, as well as mature CD4 or CD8 cells. Transgene-expressing CD4 T cells recovered from the Thy/Liv implant demonstrated resistance to vital infection in vitro. It will be important to determine whether in vitro challenge will select for the progeny of transduced stem/progenitor cells, and whether sufficient numbers of these progeny can be produced and exported to the periphery to reconstitute immune responses. -Non lentivirus models Murine leukemia virus (MuLV) Murine AIDS (MAIDS) and human AIDS exhibit common clinical features, namely severe immunodeficiency involving both T and B lymphocyte functions, enhanced susceptibility to infections, polyclonal B cell proliferation manifested by lymphadenopathy, splenomegaly and hypergammaglobulinaemia and terminal B cell lymphomas. Murine AIDS is rapidly induced within 8 to 12 weeks and mice die within 6 months post infection. Severe MAIDS is not cause by a lentivirus, but by murine leukaemia viruses (MuLV) shown to be replication-defective due to large deletions in pol and almost complete deletions in env. The 4.8 kb genomes of LP-BM5 and Duplan strains of MuLV require replication-competent ecotropic MuLV helper strains to realize their pathogenic potential. Infection with helper viruses alone does not induce disease, whereas injection of helper and defective variant viruses together causes immunodeficiency. Both radiation-induced LP-BM5 and Duplan strains possess a well-conserved gag region and code, in addition to a Pr65gag, for an unusual 60 kDa gag and a divergent p12 clevage product (Du5H) that are assumed to be important in the genesis of MAIDS. The defective variants accumulate in diseased tissues. It is interesting to note that continuous depletion of mature B cells from birth completely inhibited development of the phenotypic and functional alterations characteristic of T cells in MAIDS. Such a crucial contribution of B lymphocytes to T cell dysfunction could and should also be tested in other appropriate animal models, for example in feline and simian AIDS. The similarities of pathogenic MuLV variants to immunosuppressive and also defective feline leukaemia virus strains are remarkable. In feline leukaemia virus (FeLV) induced FAIDS, however, it is the envelop glycoprotein of the defective genome that was shown to carry the determinants of disease. The murine and feline oncoviruses are particularly suited to define precisely the gene segments responsible for disease induction, for example by recombinational insertion of small sequences from the defective pathogenic strains into the genetic background of replication-competent non-pathogenic helper viruses. Replication-defective immunodeficiency-inducing HIV variants accumulating, for example, in bone marrow or lymphopoietic tissue, have not yet been found, but one has to keep in 21 Pornpat Intarasunanont mind that standard HIV isolation procedures select for replication-competent strains only. The MuLV model provides a very cost-effective, rapid and quantitative system for the study of retrovirus-induced immunodeficiency diseases and for testing candidate antiviral agents inhibiting common retroviral function such as reverse transcription and precursor protein processing. The model will miss, however, agents targeted against special lentivirus genes such as tat, rev, nef, vpr, vpu,vpx and vif. MAIDS is also a suitable model for the study of genetic resistance to retroviral immunodeficiencies, as infection and disease development in inbred laboratory mouse strains like C57B1/6 appear to depend on H-2D and FV-1n susceptibility genes. Feline models -Lentivirus model Feline immunodeficiency virus (FIV) FIV has been recognized as a relatively common infection of pet cats (Felis catus) worldwide, although natural and vertical transmission between animals in cattery appears to be rare, reflecting low virus titres in blood, plasma and saliva. The disease manifestations are somewhat different from the consequences of HIV and SIV infections, as chronic oral cavity and upper respiratory tract infections dominate. Lymphadenopathy, anaemia, chronic enteritis and conjunctivitis and involvement of the central nervous system are also observed, as are lymphosarcomas and myeloproliferative diseases occurring late during disease progression. T and B cell responses to mitogens and antigens are reduced and CD4+ lymphocyte numbers are diminished, whereas the CD8+ and B cell populations remain stable. In contrast, T cell lymphopenia in chronically FeLV-infected cats is a results of a decrease in both CD4+ and CD8+ populations, with the corresponding ratio remaining within the normal range. FIV is antigenically unique, as sera from infected cats do not recognize antigens in Western blots of HIV-1, HIV-2, SIV, caprine arthritis-encephalitis virus (CAEV) or FeLV (except possibly for p28 of CAEV and p15E of FeLV). Neutralizing antibody or cellular immunity to FIV has not been assessed or reported. Proviral infectious molecular FIV clones have been isolated. The genomic organization of FIV is typical of lentiviruses with their gag, pol and env genes of relatively defined functions and their regulatory genes in the central region. FIV posseses a long vif-related open reading frame (ORF) between pol and env and a number of (very) short ORFs adjacent and within env whose functions remain to be determined. The clones share 80-90% amino acid sequence homologies in their gag, pol and env genes and hybridize at reduced stringencies to the gag-pol genes of HIV, SIV, equine infectious anaemia virus (EIAV) and CAEV. FIV LTRs contain binding sequences for cellular transcription factors like AP, ATF and NF-kB. 22 Pornpat Intarasunanont Co-infection by FeLV and FIV leads to an increase in FIV DNA in targey tissues (but not of FeLV DNA) and to a more severe and accelerated progression of disease. The latter is also seen in FIV-positive cats co-infected with feline infectious peritonitis virus, feline herpes and feline syncytia-forming virus (an otherwise nonpathogenic foamy virus). FIV infection of domestic cats is presently developing into a very useful animal model for human AIDS. Mechanism of pathogenesis (including neuropathogenesis), experimental drug treatment and vaccination can be investigated. Specific pathogen-free (SPF) cats, viral clones infectious in vitro and in vivo, genetic probes, monoclonal antibodies to feline cell surface antigens, efficient cell cultures and antigen and antibody assay procedures are all available. Thus, the FIV infection model can be viewed as an acceptable alternative to SIV infection of macaques and HIV infection of chimpanzees and other non-human primates. -Non lentivirus model Feline leukemia virus (FeLV-FAIDS) Replication-competent common form FeLV infectious lead to slightly reduced peripheral lymphocyte counts, anaemia and only mild depression of humoral and cellular immune responses. Late after infection, leukaemias, aplastic anaemias and other tumours may develop. More recently, a naturally occurring FeLV-FAIDS variant strain was isolated that induces a fatal immunodeficiency syndrome in nearly 100% of outbred viraemic cats. FeLV-induced FAIDS is characterized by persistent FeLV infection, lymphoid depletion, opportunistic infections, diarrhoea and weight loss. In contrast to lentivirus-induced AIDS, FeLV DNA and viral antigens are readily detected in vivo. FeLV isolates have been classified into three subgroups A, B and C, corresponding to regions of their external glycoprotein (gp70) that determine (1) susceptibility to neutralization, (2) virus interference, (3) receptor specificity and (4) tropism. FeLV-A isolates are minimally pathogenic and have been found in all naturally occurring infections, whereas FeLV-B isolates are predominantly found in cats with proliferative disorders (about 40% of all infections). The relatively rare FeLV-C strain (1% of all infections) may be responsible for aplastic anaemias. Thus, FeLV infections always involve subgroup A strains and are in part mixtures of A and B, A and C or A, B and C. The rapidly lethal immunosuppressive FeLV-FAIDS strain contained a mixture of subgroups A and B strains. These common form strains were replicationcompetent, detectable at the onset of viraemia and throughout the lifetime of the animal. In addition, FeLV-FAIDS contained replication-defective variant forms, distinguishable from common forms by restriction site polymorphism in the env gene. The variant and common form proviral DNA was molecularly cloned and defective variants could be rescued by superinfection with common form virus. Common form subgroup A or B virus strains present in the original FeLVFAIDS isolate did not induce immunodeficiency disease, whereas mixtures of both common and variant forms were rapidly pathogenic, especially in young cats. It is the 23 Pornpat Intarasunanont variant forms unintegrated DNA that accumulates in high copy numbers preferentially in bone marrow and intestinal epithelium, preceding and predicting the onset of FAIDS. Sequence comparison between common and variant form DNA also indicated that variants do not arise from the common form at high frequency in vivo. Instead, variant forms appear to be relatively stable defective virus strains pre-existing in the inoculum and rapidly amplifying in specific target tissues. FeLV represents a very attractive model for the elucidation of the genetic basis of oncovirus-induced FAIDS. The data suggest that the study of possibly defective lentivirus variants in human and animal AIDS, which may be pathogenic and hidden in retrovirus mixtures, should not be neglected. Conclusion -Advantages and disadvantages between different animal models Chimpanzee Advantages -High genetic similarity to human (about 98.5%) -Chimpanzee model can be infected with HIV-1 -Good model for study in reactive immune response Disadvantages -The model rarely develops disease -It cannot be used in sufficient numbers to provide statistically significant results -The model is large in size, not easy to handle and very expensive -Chimpanzee is a highly social animal, therefore inappropriate husbandry may interfere with its biological activities. Monkeys Advantages -Many old world monkeys, e.g. Rhesus monkey (the most prevalent monkey used in biomedical research) shares about 93% of genetic homology with human. -The models can be provided in enough numbers for study. -Some monkeys can be infected with many types of SIV, HIV-2 and are able to develop disease -They are not endangered in the wild and adapt well to captive housing. Disadvantages -The results from different SIV strain cannot be reliably extrapolated From one strain to another -It is expensive, not easy to handle, large in size -The stress of animal may interfere with its biological activities. -Risks of zoonosis 24 Pornpat Intarasunanont Mice Advantages -Small size, easy to handle, inexpensive -Highly reproductive, short generation time and life span -A lot of information about the genetic makeup is available -Large numbers available in many strains -The model can be infected with HIV-1 -Useful for examination of disease processes occurring in tissues and exploring therapeutic strategies -Mice are able to deal with genetic engineering Disadvantages -The model does not have a standard human immune system -Incomplete and low number of repopulating human lymphoid cells -SCID mice do not develop AIDS-like disease -Transgenic mice cannot yet model the process of infection Cats Advantages -Not large in size and not difficult to handle -Not very expensive -Sufficient numbers are available for study -The models can be infected with FIV, FeLV and develop immunodeficiency-like syndrome -Cause nearly identical disease to HIV infection. Disadvantages -Cat’s genetic makeup is far related to human -FIV, FeLV are not closely related to HIV -Role of nef gene Many animal studies (e.g. murine transgenic model and model vaccines in monkeys) have shown that the nef gene is quite important in eliciting the pathogenic processes. Nef (negative regulatory factor) is one of the regulatory genes of HIV (27 – 34 kD myristoylated protein unique to primate lentiviruses) A functional nef gene is important for the development of high viremia and simian AIDS in SIV infected rhesus macaques. In a transgenic mouse model expression of nef protein alone when expressed under a CD4-promoter is sufficient to cause an AIDS-like disease. A critical role for nef in development of AIDS in humans is suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection are infected with viruses carrying naturally occurring nef deletions. The mechanism of nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell proteins. Despite the recent advances in research on nef, its critical cellular function and molecular mechanism of action in the pathogenesis of AIDS remain unclear. A number of observations suggest that physical and functional interactions with host cell 25 Pornpat Intarasunanont protein kinases are important for nef function. However, the relevant protein kinases in various target cells of HIV infection still await for definitive identification, posing a major challenge for future investigations. Molecular cloning of the nef-associated serine kinase activity, NAK, would seem necessary in order to conclusively address its possible involvement in the cell biology of nef. The role(s) of different isoforms of PKC as effectors of nef will also need to be carefully elucidated. On the other hand, the potential role and the downstream molecular events of the nef/Hck interaction in providing HIV-infected macrophages with a capacity to promote HIV infection also requires further experimental attention. Also, the suggestive but contradictory reports on an interaction of nef with the T cell-specific Src-kinase Lck will have to be followed-up in more detail. If indeed both Hck and Lck turn out to be important, it will be interesting to see if they serve corresponding functions, or whether nef has evolved to employ them for different purposes in these two cell types. Although the number of suggested nef-interacting host cell proteins has rapidly increased, it is likely that not all cellular partners of nef have yet been identified. In this regard, search for novel high-affinity nef-binding SH3 domaincontaining proteins in non-myeloid cells, such as T lymphocytes, poses one attractive area for further investigations. Besides host proteins involved in signal transduction, other types of novel nef-interacting proteins are also likely to be discovered, as all pieces in the puzzle regarding the nef-induced downregulation of CD4 and MHC I are probably not yet at hand. While the identification of the relevant nef-binding host cell proteins will undoubtedly accelerate the discovery of the critical cellular processes which nef modulates in order to mediate its pathogenic function, the reverse argument can also be made. A better idea of the germane nef-induced changes in cellular physiology would greatly help to decide which ones of the candidate nef-binding host cell proteins are in fact relevant. In this regard, our current level of understanding leaves a lot of room for improvement. For example, it is still unclear if the true role of nef is to inhibit or enhance the intracellular signaling mediated through the T cell receptor complex, or whether depending on circumstances both might occur, or even, if both of these effects merely represent different artifacts of ectopic nef overexpression. Given the likely role of nef as a regulator of cellular signaling pathways, it is possible that the common practice of using established cell lines to study the effects of nef on cellular physiology may not have been optimal, and study systems which would better mimic nef expression during natural HIV infection may have to be considered more carefully in the future. SIV infection of rhesus macaques has been regarded as the ultimate test for the functionality of various nef alleles, and it is indeed the model in which the requirement for nef in the pathogenesis of AIDS was first demonstrated. In this model, unlike in cell culture systems or in SCID-hu mice, the interplay between the virus and the immune system can also be assessed which (besides being important for any infectious disease) might be relevant to appropriately reveal the in vivo function of nef. The ability to examine nef mutations which inhibit the replicative potential of the virus provides another valuable experimental tool in this 26 Pornpat Intarasunanont model. However, besides the broader question of the overall similarity of simian AIDS in SIV-infected macaques compared to AIDS in HIV-infected humans (target cell populations, immunopathogenesis, etc.), there are indications suggesting that SIV nef might differ from HIV-1 nef in certain functional aspects. Such differences, even if subtle, could significantly complicate the use of this model for testing of hypotheses based on studying HIV-1 nef. The lack of complete functional correspondence of HIV-1 and SIV nef proteins is also suggested by the efforts to create a pathogenic SIV strain in which the nef gene has been transplanted from HIV-1. While such exercises involving exchanges of much larger fragments from other regions of these genomes have been quite successful, substitution of SIV nef with HIV-1 nef has proved difficult, and positive results on this have only been obtained recently. The development of such chimeric SHIV viruses represents a significant advance, and should help to reduce the possible problems arising from dissimilarity between HIV-1 and SIV nef proteins. It should be noted, however, that accumulation of approximately half a dozen amino acid changes in certain positions of the transplanted HIV-1 nef are required for adaptation of these SHIVs to become pathogenic in macaques. Besides providing an improved model for in vivo testing of the functional importance of specific mutations introduced into the HIV-1 nef, such SHIV viruses might prove valuable in evaluating possible anti-HIV therapeutic agents targeted against nef. It is probably realistic to deem that in order to ensure continuous agreeable progress in antiretroviral therapy against AIDS, new drugs against the currently targeted as well as other HIV proteins need to be developed. The demonstrated role of nef in the pathogenesis of AIDS, together with the improved understanding about the molecular details of HIV-1 nef structure and function make Nef an attractive target for anti-HIV drug development. The necessity for HIV-1 nef protein, in order to remain functional, to preserve its architecture in critical regions which interact with host cell proteins, such as the nef SH3-binding surface, present feasible submolecular targets within the nef protein, and suggest that the therapeutic effects of drugs aimed at such sites might be less prone to be overcome by mutated drug resistant viruses than is the case with the current antiretroviral therapies. Figure 3 Some possible mechanisms of nef 27 Pornpat Intarasunanont -Benefits and problems of using animal models Benefits Animal studies of toxicity, efficacy and pharmacokinetics provide valuable scientific imformation toward identifying potentially useful antiviral agents. Useful for testing how drugs/vaccines might perform in human. Helps to understand certain mechanisms of disease and therapeutic agent observed in animal model. We can induce certain desired condition(s) in animals that can not be induced in human. Problems Preclinical studies generally are inadequate to predict whether an anti viral agent will be both effective and safe when administered to humans and it is not until the agent enters clinical trail in humans that is potential can be ascertained. There are large differences in the host cell enzymes affinities for a specific antiviral compound among species. Difficulties in interspecies interpretation. Animal is not useful in some cases that the results can only be get from study in human. Special care and husbandry are required to reduce the stress of animal, especially in higher animal like monkeys and apes. -Extrapolation The prediction of human response is not only one of the most difficult affairs of our daily life, it is also the crucial point that decides success or failure of animal experiments, and is the driving force in numerous scientific and political debates on the overall significance of animal experiments for the health of Homo sapiens. Extrapolation of results from experiments on nonhuman species to human beings carries with it enough controversy for decades of research to come. Because there are differences between species and animals do not behave the same way as humans, to minimize misinterpretation of the results, the extrapolation should meet some general requirements. Taking a plurispecies approach. Most of the regulating authorities require two species in toxicology screening, one of which has to be non-rodent. This does not necessarily imply that excessive numbers of animals will be used. The uncritical use of one-species models can mean that experimental data retrospectively turn out to be invalid for extrapolation, representing real and complete waste of animals. Using more than one species is, of course, no guarantee for successful extrapolation, either. 28 Pornpat Intarasunanont Metabolic patterns and speed must match between species. Drugs and toxins exert their effect on an organism not per se but because of the way they are metabolized, the way they and their metabolites are distributed and bound in the body tissues, and how and when they are finally excreted. The metabolism of small rodents is at least several times faster than that of humans. The visceral organs that control and exert metabolism grow slower than body size as a whole. It has been found that metabolism relates to approximately the 2/3 power of total body weight (so-called metabolic body weight, i.e., body weight2/3). Experimental doses should consequently generally be calculated according to the metabolic body weight. Russian research has demonstrated that more than 100 highly diverse biological parameters (e.g. creatinine clearance, water intake, hemoglobin weight) are linearly related to body weight, regardless of mammal species. Confounding variables of metabolism must be controlled. One must be very careful about attributing to a species or strain differences that could be due to, e.g., age, diet, sex, distress, route or time of administration and sampling, dose size, diurnal variation, season of the year, or daily temperature. Experimental design and the life situation of the target species must correspond. A model cannot be separated from the experimental design itself. If the design inadequately represents the "normal" life conditions of the target species, inaccurate conclusions may be drawn, regardless of the value of the model itself. -Future prospects Most animal models have their great potential in pathogenesis, immuneresponses analysis, vaccines and therapies study. Accordingly the challenge for the future is to utilize the increasing knowledge of molecular mechanism of HIV and HIV replication to elucidate all aspects of the virus host relationships fully and thereby to understand mechanism of pathogenesis in AIDS further. 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