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HIV infection in non-human primates: the Macaca nemestrina model
Virus Research 32 (1994) 269-282
V irus Research
HIV infection in non-human primates: the Macaw nemestrina model David M. Anderson a-*, Michael B. Agy b, Douglas Bowden b, William R. Morton b, H. Denny Liggitt a a Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA b Regional Primate Research Center, University of Washington, Seattle, WA 98195, USA (Received 8 September 1993; revised 23 November 1993; accepted 23 November 1993)
Key words: HIV infection; Non-human primate; Macnca nememina 1. Historical overview Since the initial de~ription of acquired immunodeficien~ syndrome (AIDS) in 1981 (Centers for Disease Control, 19811, AIDS has become a major health concern to the world community. An impressive array of resources have been devoted to the study of the etiologic agent, clinical course, transmission, prevention, and treatment of this disease. AIDS has been shown to be the result of persistent infection with human immunodeficien~ virus (HIV) Barre-Sinoussi et al., 1983; Gallo et al., 1984). AIDS in this country and on a worldwide basis is most commonly the result of infection with HIV Type 1 (HIV-l). However, another closely-related virus, HIV-2, has also been reported to cause similar clinical symptoms of immune depression in patients in West Africa and at least one patient in the United States (Clavel et al., 1987; Kong et al., 1988; Centers for Disease Control, 1988). One increasingly impo~ant facet of the AIDS s~drome is the neurologic compromise of infected patients. The neuropathology of adult and pediatric AIDS patients has been investigated in considerable depth (Budka, 1986; Budka et al., 1987; Levy et al., 1985; Petit0 et al., 1986; Gray et al., 1988; Masliah et al., 1992; Navia et al., 1986; De Girolami et al., 1990). Despite these efforts, many critical questions concerning the pathogenesis of HIV-associated neuropatholo~ remain. Neurologic abnormahties may accompany other signs of AIDS or may precede all
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author. Fax: + 1 (206) 685-7271.
0168-1702/94/$07.~ 0 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1702(94)00013-3
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other clinical manifestations of AIDS (Price et al., 1988; Navia and Price, 1987). Infection with HIV-l can result in a variety of neurologic complications including aseptic meningitis, AIDS dementia complex, and peripheral neuropathies (Came et al., 1985; Gabuzda and Hirsch, 1987; Berger and Levy, 1993). Primary HIV encephalopathy is considered the most common primary neurologic manifestation of HIV infection in adults and children (Price et al., 198). Symptoms of neurologic irregularities appear to be particularly aggressive in pediatric patients (Burns, 1992; Belman, 1990). We wanted to develop an animal model of AIDS in which to address the questions surrounding the neuropathologic lesions found in patients infected with HIV. This report represents a description of our initial attempt to study this aspect of HIV infection in a Macaca nemestrina animal model. 1.1. Clinical features
The clinical features associated with HIV infection have been the subject of several excellent reviews (McCutchan, 1990; Kaslow et al., 1987). Following exposure to the virus, many individuals experience an acute response characterized by non-specific symptoms such as lymph node enlargement, headaches, fever, and malaise (Tindell et al., 1988; Cooper et al., 1985; Fox et al., 1987). This acute reaction can occur three to six weeks following infection and is generally short-lived. For many patients, this acute response is followed by a period of resolution of clinical signs (Medley et al., 1987). During this period, the patient may experience few or no clinical signs of HIV infection. When present, symptoms generally are limited to non-specific, transient complaints of fever, headaches, and malaise. This period of relative abatement of clinical signs can last anywhere from months to several years (Anderson and Medley, 1988). Despite the fact that patients are often clinically asymptomatic during this period, immunologic abnormalities and antibody response are maintained (Lang et al., 1989). In addition, recent findings emphasize the fact that the virus does not enter a period of latency (Embretson et al., 1993; Pantaleo et al., 1993). Rather, viral replication still occurs in selected tissues during this period. Infection of CD4 + lymphocytes results in decreased numbers and function of T-helper cells. The decrease in T-helper lymphocytes results in an inversion of the T-helper to T-suppressor cell ratio (Ho et al., 1987). This inversion is accompanied by a sharp decrease in the competency of the cell-mediated arm of the immune system (Fauci, 1988). In addition, the normal signaling performance of T-lymphocytes is interrupted. This, in turn, causes functional deficits in the humoral immunity and natural killer activity of the immune response (Antoni et al., 1990). The patient’s gradual loss of immune competency continues until additional signs of illness are acquired. These illnesses are the often the result of opportunistic infection by agents of low pathogenicity. In addition to opportunistic infections, non-specific symptoms (fatigue, weight loss, diarrhea, fever), neurologic abnormalities, or tumors can occur in a significant percentage of patients. Occurrence of these symptoms may be preceded by laboratory hematologic abnormalities such as loss of CD4 cells, leukopenia, anemia, or thrombocytopenia. The CDC has
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developed a classification system for surveillance of HIV-infected patients (Centers for Disease Control, 1987). Individuals displaying a variety of these symptoms may be classified as either AIDS-related complex (ARC) or early AIDS. Progression of either of these stages to full-blown AIDS is uniformly followed by death in a relatively short time. 1.2. Pathology Patients dying of AIDS most often die with one or more opportunisitc infections (McCutchan, 1990). Pathologic lesions within an individual patient may be caused by opportunisitc infections, tumors, activation of latent viral agents, or primary action of HIV. Some of the more common disease processes include pneumocystis pneumonia, toxoplasma encephalitis, cytomegalovirus infection, herpes simplex virus or varicella zoster virus activation, and disseminated Mycobacterium or fungal infection (Centers for Disease Control, 1987). Tumors such as Kaposi’s sarcoma and B-cell lymphoma are also recognized in HIV-infected patients (Warner and Fisher, 1986; Ziegler et al., 1984). Specific pathologic lesions are related to the type and distribution of agents within the body. Organ systems most commonly affected include the brain, gastrointestinal system, lung, skin, and lymphatic systems (Warner and Fisher, 1986; Niedt and Schinella, 19851, Although the distribution of lesions within an individual patient is variable, the broad range of pathologic findings highlights the generalized susceptibility of AIDS patients to many disease processes. Neurologic dysfunction is currently recognized as an important clinical manifestation of HIV infection (Navia et al., 1986; Janssen, et al., 1988). In fact, occasional patients die with predominantly neurologic symptoms in the absence of opportunisitc infection. Histologic analysis of CNS autopsy tissues reveals neuropathologic lesions in up to 80% of patients (Petito, 1988) Lesions commonly encountered include white matter pallor and vacuolation, microglial nodules, multinucleate giant cells (MGC), and vacuolar myelopathy (Navia et al., 1986; Price et al., 1988). White matter pallor is commonly a diffuse change accompanied by a scattered astrocytosis. The presence and distribution of microglial nodules and MGC is variable but when present they are often found in basal ganglia, pons, and brainstem. Aseptic meningitis characterized by lymphocytic infiltrate into the leptomeninges has also been identified as a neuropathologic lesion associated with early HIV-l infection (Michaels et al., 1988b). Less common neuropathologic lesions include inflammatory infiltrates in perivascular orientation and choroid plexitis (Navia et al., 1986). Also, gray matter abnormalities including loss of neurons and alterations in dendritic morphology have been reported (Gray et al., 1988, 1991; Ketzler et al., 1990; Everall et al., 1991). 2. Materials
and methods
We wanted to produce an animal model in which to study some of the questions surrounding the development of neuropathologic lesions following HIV-l infec-
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tion. To address this topic, a study was designed using three groups, each with eight Mucaca nemestrina monkeys. One group was infected with HIV-l, one with SIVh4”, and the other group served as controls. This report will focus on the HIV-l-infected and control groups. The animals in the HIV-l group were injected with both cell-associated and cell-free virus. This was accomplished by removing peripheral blood mononuclear cells (PBMCs) at twenty days of age. Cells were stimulated for three days using PHA. PBMCs were divided into three groups. Each group of PBMCs was incubated in the presence of a different strain of HIV-l for seven days. The three strains of virus were LAI, JR-FL, and JR-CSF. The LAI strain of virus is a strain of virus previously proven to persistently infect the Macaca nemesthuz species (Agy et al., 1992). The JR-FL virus is a viral isolate from the frontal lobe of an AIDS patient. Similarly, the JR-CSF virus is an isolate from the cerebral spinal fluid of the same patient. Productive infection of PBMCs was assayed using a viral antigen capture assay. Following infection of the PBMCs with HIV-l, autologous cells were injected intravenously into animals in a vehicle containing cell-free virus. All animals were injected intravenously at one month of age. Control animals were also injected with non-infected autologous PBMCs in vehicle without cell-free virus. Two animals from each group were killed at two, six, fourteen, and twenty-four weeks post-injection. All animals were clinically normal at the time of sacrifice. Animals were sacrificed via perfusion with paraformaldehyde while under Nembutal@ anesthesia. The perfusion process was accomplished using an infusion pump with flow rate adjusted for the size of the animal. Immediately prior to the perfusion process, animals were injected with 1.0 ml of 1% sodium nitrite solution for vasodilation. Animals were perfused with 1% paraformaldehyde for 1 min and then 4% paraformaldehyde for 9 min. The brain was then removed and fluid displacement was measured. The pituitary and cervical spinal cord were also removed. All tissues were placed in 4% paraformaldehyde at 4°C for 6 h following removal. After post-fixing, tissues were then placed in a 10% glycerol, 2% DMSO in PBS solution at 4°C overnight. Twelve to twenty-four hours later, tissues were processed for either paraffin embedding or cryopreservation. The right hemisphere of each brain was examined histologically. Sagittal blocks containing thalamic and hypothalamic structures were removed and embedded in paraffin. The remainder of the right hemisphere was cut into 5-mm coronal slices. These slices proceeded anterior and posterior from the anterior commissure. Alternate slices were either embedded in paraffin or processed for cryopreservation. Tissues from the pituitary and cervical spinal cord were embedded in paraffin. Cryopreservation consisted of filling an embedding mold containing tissue with OCT and freezing in isopentane cooled with liquid nitrogen. Paraffin-embedded tissues were examined for evidence of neuropathology. Blocks containing thalamic and hypothalamic structures were serial sectioned. Slides were cut at 7 Jo and every third slide was stained with hematoxylin and eosin for histologic examination. For the remainder of the paraffin blocks, ten sections
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were cut from the anterior face of each block. Sections two and seven were stained and examined. A similar procedure was used for tissues from the pituitary and multiple sections of cervical spinal cord. Unstained sections were probed for HIV-l viral antigen using DuPont@ anti-p24, gp41, and gp120 antibodies and a Vector ABC Elite@ kit according to manufacturer’s instructions. Selected sections were immunostained for glial fibrillary acidic protein (GFAP) using a monoclonal antibody obtained from Sternberger Monoclonals Inc. Host:virus interaction
The structure and life cycle of HIV-l has been reviewed by several authors (McCutchan, 1990; Haseltine and Wong-Staal, 19881. Briefly, the virus attaches and enters a susceptible cell via the CD4 membrane glycoprotein. The virus initiates reverse transcription of its RNA to form DNA which is then inserted into the genome of the host cell. Transcription of the DNA with processing to produce spliced RNA products is induced by as yet unclear mechanisms. RNA products are then translated and processed to produce viral proteins. These proteins are then encapsulated into a viral core to form an infectious virion. This is then released through the cellular membrane. A considerable amount of effort has been devoted to identification of cell types susceptible to HIV-l infection. Cells expressing the CD4 membrane glycoprotein have been shown to be readily infected with HIV (Weber and Weiss, 1988). Circulating cells readily infected include CD4 + T-helper lymphocytes and macrophages. In the central nervous system, it appears that only cells of monocyte origin are demonstrably susceptible to infection with HIV. Multinucleate giant cells, macrophages, and microglial cells have all been demonstrated to be positive for HIV infection (Wiley et al., 1986; Michaels et al., 1988a). Additional studies have suggested that endothelial cells (Rostad et al., 1987; Rhodes, 19911, astrocytes (Mirra and de1 Rio, 19891, neurons (Staler et al., 19761, and oligodendrocytes (Gyorkey et al., 1987) are also susceptible to infection. However, these findings have been challenged by others and currently can be viewed as equivocal (Price et al., 1988; Budka, 1990; Budka and Lassman, 1988; Kure et al., 1990; Sharer and Prineas, 1988). The mechanism for entry of HIV into the CNS remains unclear at this time. Several hypotheses have been suggested by different authors. Currently, the most probable theory proposes that the virus enters the CNS via the bloodstream through infected monocytes. Despite the lack of direct information regarding the pathogenesis of viral entry into the CNS, it is clear that virus enters early in infection (Michaels et al., 1988; Davis et al., 1991). Once inside the brain and CSF compartments, HIV is associated with development of neuropathologic lesions through as yet undefined mechanisms. 3. Results The neuropathologic lesions associated with HIV infection have been described above. The pilot project summarized in this report was designed to identify
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Table 1 Neuropathology of HIV-1 infected macaques
2 Week 592146 K92136 6 Week 591350 591358 14 Week F92198 F92199 24 Week F91364 91367
perivascular cuffing
Microglial nodules
-
_
+ (severe) -
+ (rare) _
_ -
_
_ _
_
Choroid Plexitis
Ependymal denudation
+ (mild) + (mild)
Meningitis
_ +
+ (mild)
_ _
+ (mild) + (mild)
+ _ + (severe)
-
+ (mild)
histologic lesions in the CNS of HIV-l-infected pigtailed macaques. The histologic findings in the CNS are outlined in Table 1. Five out of eight animals examined displayed some type of neurologic lesions within the right hemisphere. One animal killed at 2 weeks PI, both animals at 6 weeks PI, one animal at 14 weeks PI, and one animal at 24 weeks PI had histologic lesions in the right hemisphere. Histologic lesions include perivascular cuffing (Fig. 11, microglial nodules (Fig. 2), choroid plexitis (Fig. 3), ependymal denudation, and meningitis. The perivascular cuffing in animal 591350 and the infiltrate into the choroid plexus of animal F91364 are judged to be severe. Lesions found in other animals are mild in severity. The perivascular infiltrate present in animal F91350 was localized to the gray-white junction in the area of the occipital cortex. The cellular infiltrate surrounds the vascular branches as they enter a rather narrow zone at this junction
Fig. 1. Perivascular cuffing, animal 591350, H&E, 200x. junction in occipital cortex.
Perivascular cuffing limited to gray-white
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Fig. 2. Microglial nodule (arrow), animal 591350, H&E, 400 X . Rare microglial nodules were present in the basal ganglia.
and diminish as the vessel leaves this area. The microglial nodule pictured (Fig. 2) was found in the basal ganglia of animal 591350. An inflammatory cell infiltrate in the choroid plexus was present in animals 591350,591358, and F91364. Denudation of ependymal cells was randomly distributed in the lateral ventricles of animals K92136 and F92198. Cellular infiltrates into the leptomeninges in animals K92136, 591350, 591358, and F91364 also did not show a predisposition for any region. The cellular infiltrates into the choroid plexus and meninges appear to be predominantly lymphocytic with occasional plasma cells present. The perivascular cuffing is composed of a mixed population including macrophages, lymphocytes, astrocytes, and occasional plasma cells. Immunostaining for glial fibrillary acidic protein (GFAP) reveals a marked increase in staining in areas adjacent to perivas-
Fig. 3. Choroid plexitis, animal 591364, H&E, 400 X . Choroid plexitis was present ventricle. Similar lesions were also present in animals 591350 and 591358.
in the lateral
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Fig. 4. GFAP immunostaining of perivascular cuffing, animal 591350, 82X. A: Age;matched control animal. No staining outside of white matter. B: Animal J913.50. Moderate positive staining indicates astrocytosis in area immediately surrounding perivascular cuffing. C: Animal 5913.50. Marked positive staining at gray-white junction associated with marked astrocytosis in area of perivascular cuffing.
cular cuffs (Fig. 4 A-C). Immunostaining with CFAP revealed no evidence of diffuse astrocytosis. No histologic lesions were recognized in sections of pituitary or cervical spinal cord. Immunostaining for viral antigen was negative for all animals. No age predisposition towards lesion type, severity, or distribution is appreciated. White matter abnormalities were equivocal. Mild pallor and vacuolation were not confirmed by Luxol-fast blue staining or the presence of diffuse astrocytosis or maerophages. No histologic Iesions were found in control animals.
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4. Discussion
These findings indicate that histologic lesions in the central nervous system are temporally associated with HIV-l infection of infant Mucaca nemestrina monkeys. The overall distribution of lesions is similar to that found on examination of CNS tissues from human AIDS patients. Histologic examination of autopsy tissues from AIDS patients display neuropathologic lesions in up to 80% of cases (Petito, 1988). These changes include white matter abnormalities, microglial nodules, multinucleate giant cells, meningitis, perivascular lymphoid infiltrations, choroid plexitis, reduced numbers of neurons, and dendritic abnormalities (Budka, 1986; Budka et al, 1987; Levy et al., 1985; Petit0 et al., 1986; Gray et al., 1988; Masliah et al., 1992; Navia et al., 1986; De Girolami et al., 1990). Many of these histologic changes, including lesion distribution, are also present in this animal model. For instance, four of the animals displayed meningitis and three of these animals also displayed concomitant choroid plexitis. Also, one animal had severe perivascular cuffing and rare microglial nodules. White matter abnormalities were judged to be equivocal in the brains examined. Despite a comprehensive analysis of CNS tissues, no MGCs were recognized in any of the brains examined. Despite the presence of lesions in the CNS, the significance of these lesions remains equivocal. In most animals, the lesions are mild and non-specific in nature. It is unclear whether the lesions present indicate infection of the CNS or a response to a systemic viral infection. It may be that the relatively mild lesions are merely the result of a shortened time course for disease progression. Previous studies of autopsy material from individuals infected with HIV-l but asymptomatic revealed increased numbers of perivascular lymphocytic cells but greatly decreased lesions generally associated with HIV (Esiri et al., 1989). It may be postulated that the neuropathologic lesions commonly associated with HIV infection in humans may develop over a considerable length of time. Animals allowed to survive for a time comparable to human patients may develop more severe CNS lesions. Conversely, it may be postulated that the lesions are not indicative of direct infection of the CNS. The lesions might be mediated through secondary messengers, i.e., cytokines, responding to the systemic viral infection. Studies have demonstrated increased levels of circulating cytokines such as tumor necrosis factor and interleukin-1 in HIV-infected individuals (Lahdevirta et al., 1988; Lepe-Zuniga et al., 1987). Circulating or local cytokine production may be the underlying factor in the pathogensis of neuropathologic lesions without direct infection of the CNS. The inability to demonstrate viral antigen using immunocytochemistry and the lack of multinucleate giant cells leave unresolved the question of whether the virus actually infects CNS tissues in this model. In order to resolve this question, demonstration of viral presence in the CNS is necessary. This might be accomplished by virus isolation, demonstration of intrathecal synthesis of antibodies to viral antigens, or demonstration of HIV sequences using in situ hybridization or polymerase chain reaction techniques. Until virus or viral antigen can be demonstrated in central nervous tissues, the pathogenesis of histologic lesions remains speculative.
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Comparison with other lentivirual infections Relatively few animal models using HIV-l are currently available to the research community. Non-human primates such as chimps (Pan troglodytes) and gibbons (Hylobates lar) have previously been found to be susceptible to persistent infection with HIV-l as evidenced by development of high titer virus-specific antibody responses following infection (Lusso et al., 1988; Alter et al., 1984). However, neither or these species develops clinical signs of infection or pathologic lesions (Watanabe et al., 1991). The endangered status of these species further limits the utilization of these models. Although the modeling of HIV-l infection in these species may offer some utility in vaccine development research, the limitations of these models preclude their use in many areas of HIV investigation. An alternative animal model of HIV-1 is the severe combined immunodeficient @CID) mouse reconstitued with human cells, human fetal liver cells, orhuman bone marrow (Mosier et al., 1988; McCune et al., 1988; Lubin et al., 1991). A SCID mouse reconstituted with human peripheral blood lymphocytes (hu-PBLSCID) has been developed which is susceptible to infection with HIV-l isolates (Mosier et al., 1991). These models may prove useful in addressing questions related to depletion of CD4 + T-lymphocytes in HIV-infected patients. However, the lack of correlation between in vivo pathogenicity and in vitro properties appears to limit the usefulness of these models for investigation of pathogenic potential in vivo (Mosier et al., 1993). In summary, it appears that the HIV-l infected Macaca nemestrina may represent a new animal model for aspects of human HIV infection. Animals inoculated with these strains of HIV-l become persistently infected and display evidence of neuropathologic lesions in the CNS4’. Although virus has not been demonstrated in central nervous tissues, these data indicate that infection is temporally associated with development of lesions. As such, this model may prove useful for future studies in HIV pathogenesis, treatment, vaccine development, and maternal-fetal transmission. These prelimary data suggest that this model may become a useful tool in the continuing efforts to develop effective measures to combat the worldwide AIDS epidemic. Acknowledgements
The authors would like to thank Dr. Clayton Wiley and Dr. Carol Petit0 for their immunochemical examination of tissue sections. We thank Dr. Mark Sumi for his assistance in neuropathologic evaluation of tissues. We would also like to thank Charlene Karr-May for her technical assistance. This work was supported by grants USPHS RR00166 and Comparative Medicine Training Grant RR07019. References Agy, M.B., Frumkin, L.R., Corey, L., Coombs, R.W., Wolinsky, S.M., Koehler, J., Morton, W.R. and Katze, M.G. (1992) Infection of Macaca nemestrina by human immunodeficiency virus type-l. Science 257, 103-6.
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McCune, J.M., Namikawa, R., Kaneshima, H., Shultz, L.D., Lieberman, M. and Weissman, I.L. (1988) The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241, 1632. Medley, G.F., Anderson, R.M., Cox, D.R. and Billard, L. (1987) Incubation period of AIDS in patients infected via blood transfusion. Nature 328, 719-21. Michaels, J., Price, R.W. and Rosenblum, M.K. (1988a) Microglia in the giant cell encephalitis of acquired immune deficiency syndrome: proliferation, infection, and fusion. Acta Neuropathol. 76, 373-9. Michaels, J., Sharer, L.R. and Epstein, LG. (1988b) Human immunodeficiency virus type 1 (HIV) infection of the nervous system: a review. Immunodef Rev. 1, 71-104. Mirra, S.S. and de1 Rio, C. (1989) The fine structure of acquired immunodeficiency syndrome encephalopathy. Arch. Pathol. Lab. Med. 113, 858-65. Mosier, D.E., Gulizia, R.J., Baird, S.M. and Wilson, D.B. (1988) Transfer of a functional humaniImmune system to mice with severe combined immunodeficiency. Nature 335, 256. Mosier, D.E., Gulizia, R.J., Baird, SM., Wilson, D.B., Spector, D.H. and Spector, S.A. (1991) Human immunodeficiency virus infection of human-PBL-SCID mice. Science 251, 791-94. Mosier, D.E., Gulizia, R.J., MacIsaac, P.D., Torbett, B.E. and Levy, J.A. (1993) Rapid loss of CD4+ T-cells in human-PBL-SCID mice by noncytopathic HIV isolates. Science 260, 689-92. Navia, B.A. and Price, R.W. (1987) The acquired immunodeficiency syndrome dementia complex as the presenting or sole manifestation of human immunodeficiency virus infection. Arch. Neurol. 44, 65-9. Navia, B.A., Cho E-S., Petito, C.K. and Price, R.W. (1986) The AIDS dementia complex: II. Neuropatholgy. Ann. Neurol. 19, 525-35. Niedt, G.W. and Schinella, R.A. (1985) Acquired immunodeficiency syndrome: clinicopathologic study of 56 autopsies. Arch. Pathol. Lab. Med. 109, 727-34. Pantaleo, G., Graziosi, C., Demarest, J.F., Butini, L., Montroni, M., Fox, C.H., Orenstein, J.M., Kotler, D.P. and Fauci, A.S. (1993) HIV infection is active and progressive in lymphoid tissue during he clinically latent stage of disease. Nature 362, 355-58. Petito, C.K. (1988) Review of central nervous system pathology in human immunodeficiency virus infection. Ann. Neurol. 19, 517-524. Petito, C.K., Cho E-S., Lemann, W., Navia, B.A. and Price, R.W. (1986) Neuropathology Of acquired immunodeficiency syndrome (AIDS): an autopsy review. J. Neuropathol. Exp. Neurol. 45, 635-646. Price, R.W., Brew, B., Sidtis, J., Rosenblum, M., Scheck, A.C. and Cleary, P. (1988) The brain in AIDS: central nervous system HIV-l infection and AIDS dementia complex. Science 239, 586-592. Rhodes, R.H. (1991) Evidence of serum-protein leakage across the blood-brain barrier in the acquired immunodeficiency syndrome. J. Neuropathol. Exp. Neurol. 50, 171-83. Rostad, SW., Sumi, S.M., Shaw C-M., Olson, K. and McDougall, J.K. (1987) Human immunodeficiencey virus (HIV) infection in brains with AIDS-related leukoencephalopathy. AIDS Res. Hum. Retro 3, 363-73. Sharer, L.R. and Prineas, J.W. (1988) Human immunodeficiency virus in glial cells, continued (letter). J. Infec. Dis. 157, 204. Stoler, M.H., Eskin, T.A., Benn, S., Angerer, R.C. and Angerer, L.M. (1976) Human T-cell lymphotropic virus type III infection of the central nervous system: a preliminary in situ analysis. JAMA 256, 2360-4. Tindell, B., Barker, S., Donovan, B., Barnes, T., Roberts, J., Kronenberg, C., Gold, J., Penny, R., Cooper, D. and The Sydney AIDS Study Group (1988) Characterization of the acute illness associated with human immunodeficiency virus infection. Arch. Internal. Med. 148, 945-49. Warner, L.C. and Fisher, B.K. (1986) Cutaneous manifestations of the acquired immunodeficiency syndrome. Int. J. Dennatol. 25, 337-50. Watanabe, M., Ringler, D.J., Fultz, P.N., MacKey J.J., Boyson, J.E., Levine, C.G. and Letvin, N.L. (1991) A chimpanzee-passaged human immunodeficiency virus isolate is cytopathic for chimpanzee cells but does not induce disease. J. Virol. 65, 3344-3348. Weber, J. and Weiss, R.A. (1988) HIV Infection: the cellular picture. Sci. Am. 259, 101-109.
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V irus Research
HIV infection in non-human primates: the Macaw nemestrina model David M. Anderson a-*, Michael B. Agy b, Douglas Bowden b, William R. Morton b, H. Denny Liggitt a a Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA b Regional Primate Research Center, University of Washington, Seattle, WA 98195, USA (Received 8 September 1993; revised 23 November 1993; accepted 23 November 1993)
Key words: HIV infection; Non-human primate; Macnca nememina 1. Historical overview Since the initial de~ription of acquired immunodeficien~ syndrome (AIDS) in 1981 (Centers for Disease Control, 19811, AIDS has become a major health concern to the world community. An impressive array of resources have been devoted to the study of the etiologic agent, clinical course, transmission, prevention, and treatment of this disease. AIDS has been shown to be the result of persistent infection with human immunodeficien~ virus (HIV) Barre-Sinoussi et al., 1983; Gallo et al., 1984). AIDS in this country and on a worldwide basis is most commonly the result of infection with HIV Type 1 (HIV-l). However, another closely-related virus, HIV-2, has also been reported to cause similar clinical symptoms of immune depression in patients in West Africa and at least one patient in the United States (Clavel et al., 1987; Kong et al., 1988; Centers for Disease Control, 1988). One increasingly impo~ant facet of the AIDS s~drome is the neurologic compromise of infected patients. The neuropathology of adult and pediatric AIDS patients has been investigated in considerable depth (Budka, 1986; Budka et al., 1987; Levy et al., 1985; Petit0 et al., 1986; Gray et al., 1988; Masliah et al., 1992; Navia et al., 1986; De Girolami et al., 1990). Despite these efforts, many critical questions concerning the pathogenesis of HIV-associated neuropatholo~ remain. Neurologic abnormahties may accompany other signs of AIDS or may precede all
* Corresponding
author. Fax: + 1 (206) 685-7271.
0168-1702/94/$07.~ 0 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1702(94)00013-3
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other clinical manifestations of AIDS (Price et al., 1988; Navia and Price, 1987). Infection with HIV-l can result in a variety of neurologic complications including aseptic meningitis, AIDS dementia complex, and peripheral neuropathies (Came et al., 1985; Gabuzda and Hirsch, 1987; Berger and Levy, 1993). Primary HIV encephalopathy is considered the most common primary neurologic manifestation of HIV infection in adults and children (Price et al., 198). Symptoms of neurologic irregularities appear to be particularly aggressive in pediatric patients (Burns, 1992; Belman, 1990). We wanted to develop an animal model of AIDS in which to address the questions surrounding the neuropathologic lesions found in patients infected with HIV. This report represents a description of our initial attempt to study this aspect of HIV infection in a Macaca nemestrina animal model. 1.1. Clinical features
The clinical features associated with HIV infection have been the subject of several excellent reviews (McCutchan, 1990; Kaslow et al., 1987). Following exposure to the virus, many individuals experience an acute response characterized by non-specific symptoms such as lymph node enlargement, headaches, fever, and malaise (Tindell et al., 1988; Cooper et al., 1985; Fox et al., 1987). This acute reaction can occur three to six weeks following infection and is generally short-lived. For many patients, this acute response is followed by a period of resolution of clinical signs (Medley et al., 1987). During this period, the patient may experience few or no clinical signs of HIV infection. When present, symptoms generally are limited to non-specific, transient complaints of fever, headaches, and malaise. This period of relative abatement of clinical signs can last anywhere from months to several years (Anderson and Medley, 1988). Despite the fact that patients are often clinically asymptomatic during this period, immunologic abnormalities and antibody response are maintained (Lang et al., 1989). In addition, recent findings emphasize the fact that the virus does not enter a period of latency (Embretson et al., 1993; Pantaleo et al., 1993). Rather, viral replication still occurs in selected tissues during this period. Infection of CD4 + lymphocytes results in decreased numbers and function of T-helper cells. The decrease in T-helper lymphocytes results in an inversion of the T-helper to T-suppressor cell ratio (Ho et al., 1987). This inversion is accompanied by a sharp decrease in the competency of the cell-mediated arm of the immune system (Fauci, 1988). In addition, the normal signaling performance of T-lymphocytes is interrupted. This, in turn, causes functional deficits in the humoral immunity and natural killer activity of the immune response (Antoni et al., 1990). The patient’s gradual loss of immune competency continues until additional signs of illness are acquired. These illnesses are the often the result of opportunistic infection by agents of low pathogenicity. In addition to opportunistic infections, non-specific symptoms (fatigue, weight loss, diarrhea, fever), neurologic abnormalities, or tumors can occur in a significant percentage of patients. Occurrence of these symptoms may be preceded by laboratory hematologic abnormalities such as loss of CD4 cells, leukopenia, anemia, or thrombocytopenia. The CDC has
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developed a classification system for surveillance of HIV-infected patients (Centers for Disease Control, 1987). Individuals displaying a variety of these symptoms may be classified as either AIDS-related complex (ARC) or early AIDS. Progression of either of these stages to full-blown AIDS is uniformly followed by death in a relatively short time. 1.2. Pathology Patients dying of AIDS most often die with one or more opportunisitc infections (McCutchan, 1990). Pathologic lesions within an individual patient may be caused by opportunisitc infections, tumors, activation of latent viral agents, or primary action of HIV. Some of the more common disease processes include pneumocystis pneumonia, toxoplasma encephalitis, cytomegalovirus infection, herpes simplex virus or varicella zoster virus activation, and disseminated Mycobacterium or fungal infection (Centers for Disease Control, 1987). Tumors such as Kaposi’s sarcoma and B-cell lymphoma are also recognized in HIV-infected patients (Warner and Fisher, 1986; Ziegler et al., 1984). Specific pathologic lesions are related to the type and distribution of agents within the body. Organ systems most commonly affected include the brain, gastrointestinal system, lung, skin, and lymphatic systems (Warner and Fisher, 1986; Niedt and Schinella, 19851, Although the distribution of lesions within an individual patient is variable, the broad range of pathologic findings highlights the generalized susceptibility of AIDS patients to many disease processes. Neurologic dysfunction is currently recognized as an important clinical manifestation of HIV infection (Navia et al., 1986; Janssen, et al., 1988). In fact, occasional patients die with predominantly neurologic symptoms in the absence of opportunisitc infection. Histologic analysis of CNS autopsy tissues reveals neuropathologic lesions in up to 80% of patients (Petito, 1988) Lesions commonly encountered include white matter pallor and vacuolation, microglial nodules, multinucleate giant cells (MGC), and vacuolar myelopathy (Navia et al., 1986; Price et al., 1988). White matter pallor is commonly a diffuse change accompanied by a scattered astrocytosis. The presence and distribution of microglial nodules and MGC is variable but when present they are often found in basal ganglia, pons, and brainstem. Aseptic meningitis characterized by lymphocytic infiltrate into the leptomeninges has also been identified as a neuropathologic lesion associated with early HIV-l infection (Michaels et al., 1988b). Less common neuropathologic lesions include inflammatory infiltrates in perivascular orientation and choroid plexitis (Navia et al., 1986). Also, gray matter abnormalities including loss of neurons and alterations in dendritic morphology have been reported (Gray et al., 1988, 1991; Ketzler et al., 1990; Everall et al., 1991). 2. Materials
and methods
We wanted to produce an animal model in which to study some of the questions surrounding the development of neuropathologic lesions following HIV-l infec-
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tion. To address this topic, a study was designed using three groups, each with eight Mucaca nemestrina monkeys. One group was infected with HIV-l, one with SIVh4”, and the other group served as controls. This report will focus on the HIV-l-infected and control groups. The animals in the HIV-l group were injected with both cell-associated and cell-free virus. This was accomplished by removing peripheral blood mononuclear cells (PBMCs) at twenty days of age. Cells were stimulated for three days using PHA. PBMCs were divided into three groups. Each group of PBMCs was incubated in the presence of a different strain of HIV-l for seven days. The three strains of virus were LAI, JR-FL, and JR-CSF. The LAI strain of virus is a strain of virus previously proven to persistently infect the Macaca nemesthuz species (Agy et al., 1992). The JR-FL virus is a viral isolate from the frontal lobe of an AIDS patient. Similarly, the JR-CSF virus is an isolate from the cerebral spinal fluid of the same patient. Productive infection of PBMCs was assayed using a viral antigen capture assay. Following infection of the PBMCs with HIV-l, autologous cells were injected intravenously into animals in a vehicle containing cell-free virus. All animals were injected intravenously at one month of age. Control animals were also injected with non-infected autologous PBMCs in vehicle without cell-free virus. Two animals from each group were killed at two, six, fourteen, and twenty-four weeks post-injection. All animals were clinically normal at the time of sacrifice. Animals were sacrificed via perfusion with paraformaldehyde while under Nembutal@ anesthesia. The perfusion process was accomplished using an infusion pump with flow rate adjusted for the size of the animal. Immediately prior to the perfusion process, animals were injected with 1.0 ml of 1% sodium nitrite solution for vasodilation. Animals were perfused with 1% paraformaldehyde for 1 min and then 4% paraformaldehyde for 9 min. The brain was then removed and fluid displacement was measured. The pituitary and cervical spinal cord were also removed. All tissues were placed in 4% paraformaldehyde at 4°C for 6 h following removal. After post-fixing, tissues were then placed in a 10% glycerol, 2% DMSO in PBS solution at 4°C overnight. Twelve to twenty-four hours later, tissues were processed for either paraffin embedding or cryopreservation. The right hemisphere of each brain was examined histologically. Sagittal blocks containing thalamic and hypothalamic structures were removed and embedded in paraffin. The remainder of the right hemisphere was cut into 5-mm coronal slices. These slices proceeded anterior and posterior from the anterior commissure. Alternate slices were either embedded in paraffin or processed for cryopreservation. Tissues from the pituitary and cervical spinal cord were embedded in paraffin. Cryopreservation consisted of filling an embedding mold containing tissue with OCT and freezing in isopentane cooled with liquid nitrogen. Paraffin-embedded tissues were examined for evidence of neuropathology. Blocks containing thalamic and hypothalamic structures were serial sectioned. Slides were cut at 7 Jo and every third slide was stained with hematoxylin and eosin for histologic examination. For the remainder of the paraffin blocks, ten sections
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were cut from the anterior face of each block. Sections two and seven were stained and examined. A similar procedure was used for tissues from the pituitary and multiple sections of cervical spinal cord. Unstained sections were probed for HIV-l viral antigen using DuPont@ anti-p24, gp41, and gp120 antibodies and a Vector ABC Elite@ kit according to manufacturer’s instructions. Selected sections were immunostained for glial fibrillary acidic protein (GFAP) using a monoclonal antibody obtained from Sternberger Monoclonals Inc. Host:virus interaction
The structure and life cycle of HIV-l has been reviewed by several authors (McCutchan, 1990; Haseltine and Wong-Staal, 19881. Briefly, the virus attaches and enters a susceptible cell via the CD4 membrane glycoprotein. The virus initiates reverse transcription of its RNA to form DNA which is then inserted into the genome of the host cell. Transcription of the DNA with processing to produce spliced RNA products is induced by as yet unclear mechanisms. RNA products are then translated and processed to produce viral proteins. These proteins are then encapsulated into a viral core to form an infectious virion. This is then released through the cellular membrane. A considerable amount of effort has been devoted to identification of cell types susceptible to HIV-l infection. Cells expressing the CD4 membrane glycoprotein have been shown to be readily infected with HIV (Weber and Weiss, 1988). Circulating cells readily infected include CD4 + T-helper lymphocytes and macrophages. In the central nervous system, it appears that only cells of monocyte origin are demonstrably susceptible to infection with HIV. Multinucleate giant cells, macrophages, and microglial cells have all been demonstrated to be positive for HIV infection (Wiley et al., 1986; Michaels et al., 1988a). Additional studies have suggested that endothelial cells (Rostad et al., 1987; Rhodes, 19911, astrocytes (Mirra and de1 Rio, 19891, neurons (Staler et al., 19761, and oligodendrocytes (Gyorkey et al., 1987) are also susceptible to infection. However, these findings have been challenged by others and currently can be viewed as equivocal (Price et al., 1988; Budka, 1990; Budka and Lassman, 1988; Kure et al., 1990; Sharer and Prineas, 1988). The mechanism for entry of HIV into the CNS remains unclear at this time. Several hypotheses have been suggested by different authors. Currently, the most probable theory proposes that the virus enters the CNS via the bloodstream through infected monocytes. Despite the lack of direct information regarding the pathogenesis of viral entry into the CNS, it is clear that virus enters early in infection (Michaels et al., 1988; Davis et al., 1991). Once inside the brain and CSF compartments, HIV is associated with development of neuropathologic lesions through as yet undefined mechanisms. 3. Results The neuropathologic lesions associated with HIV infection have been described above. The pilot project summarized in this report was designed to identify
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Table 1 Neuropathology of HIV-1 infected macaques
2 Week 592146 K92136 6 Week 591350 591358 14 Week F92198 F92199 24 Week F91364 91367
perivascular cuffing
Microglial nodules
-
_
+ (severe) -
+ (rare) _
_ -
_
_ _
_
Choroid Plexitis
Ependymal denudation
+ (mild) + (mild)
Meningitis
_ +
+ (mild)
_ _
+ (mild) + (mild)
+ _ + (severe)
-
+ (mild)
histologic lesions in the CNS of HIV-l-infected pigtailed macaques. The histologic findings in the CNS are outlined in Table 1. Five out of eight animals examined displayed some type of neurologic lesions within the right hemisphere. One animal killed at 2 weeks PI, both animals at 6 weeks PI, one animal at 14 weeks PI, and one animal at 24 weeks PI had histologic lesions in the right hemisphere. Histologic lesions include perivascular cuffing (Fig. 11, microglial nodules (Fig. 2), choroid plexitis (Fig. 3), ependymal denudation, and meningitis. The perivascular cuffing in animal 591350 and the infiltrate into the choroid plexus of animal F91364 are judged to be severe. Lesions found in other animals are mild in severity. The perivascular infiltrate present in animal F91350 was localized to the gray-white junction in the area of the occipital cortex. The cellular infiltrate surrounds the vascular branches as they enter a rather narrow zone at this junction
Fig. 1. Perivascular cuffing, animal 591350, H&E, 200x. junction in occipital cortex.
Perivascular cuffing limited to gray-white
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Fig. 2. Microglial nodule (arrow), animal 591350, H&E, 400 X . Rare microglial nodules were present in the basal ganglia.
and diminish as the vessel leaves this area. The microglial nodule pictured (Fig. 2) was found in the basal ganglia of animal 591350. An inflammatory cell infiltrate in the choroid plexus was present in animals 591350,591358, and F91364. Denudation of ependymal cells was randomly distributed in the lateral ventricles of animals K92136 and F92198. Cellular infiltrates into the leptomeninges in animals K92136, 591350, 591358, and F91364 also did not show a predisposition for any region. The cellular infiltrates into the choroid plexus and meninges appear to be predominantly lymphocytic with occasional plasma cells present. The perivascular cuffing is composed of a mixed population including macrophages, lymphocytes, astrocytes, and occasional plasma cells. Immunostaining for glial fibrillary acidic protein (GFAP) reveals a marked increase in staining in areas adjacent to perivas-
Fig. 3. Choroid plexitis, animal 591364, H&E, 400 X . Choroid plexitis was present ventricle. Similar lesions were also present in animals 591350 and 591358.
in the lateral
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Fig. 4. GFAP immunostaining of perivascular cuffing, animal 591350, 82X. A: Age;matched control animal. No staining outside of white matter. B: Animal J913.50. Moderate positive staining indicates astrocytosis in area immediately surrounding perivascular cuffing. C: Animal 5913.50. Marked positive staining at gray-white junction associated with marked astrocytosis in area of perivascular cuffing.
cular cuffs (Fig. 4 A-C). Immunostaining with CFAP revealed no evidence of diffuse astrocytosis. No histologic lesions were recognized in sections of pituitary or cervical spinal cord. Immunostaining for viral antigen was negative for all animals. No age predisposition towards lesion type, severity, or distribution is appreciated. White matter abnormalities were equivocal. Mild pallor and vacuolation were not confirmed by Luxol-fast blue staining or the presence of diffuse astrocytosis or maerophages. No histologic Iesions were found in control animals.
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4. Discussion
These findings indicate that histologic lesions in the central nervous system are temporally associated with HIV-l infection of infant Mucaca nemestrina monkeys. The overall distribution of lesions is similar to that found on examination of CNS tissues from human AIDS patients. Histologic examination of autopsy tissues from AIDS patients display neuropathologic lesions in up to 80% of cases (Petito, 1988). These changes include white matter abnormalities, microglial nodules, multinucleate giant cells, meningitis, perivascular lymphoid infiltrations, choroid plexitis, reduced numbers of neurons, and dendritic abnormalities (Budka, 1986; Budka et al, 1987; Levy et al., 1985; Petit0 et al., 1986; Gray et al., 1988; Masliah et al., 1992; Navia et al., 1986; De Girolami et al., 1990). Many of these histologic changes, including lesion distribution, are also present in this animal model. For instance, four of the animals displayed meningitis and three of these animals also displayed concomitant choroid plexitis. Also, one animal had severe perivascular cuffing and rare microglial nodules. White matter abnormalities were judged to be equivocal in the brains examined. Despite a comprehensive analysis of CNS tissues, no MGCs were recognized in any of the brains examined. Despite the presence of lesions in the CNS, the significance of these lesions remains equivocal. In most animals, the lesions are mild and non-specific in nature. It is unclear whether the lesions present indicate infection of the CNS or a response to a systemic viral infection. It may be that the relatively mild lesions are merely the result of a shortened time course for disease progression. Previous studies of autopsy material from individuals infected with HIV-l but asymptomatic revealed increased numbers of perivascular lymphocytic cells but greatly decreased lesions generally associated with HIV (Esiri et al., 1989). It may be postulated that the neuropathologic lesions commonly associated with HIV infection in humans may develop over a considerable length of time. Animals allowed to survive for a time comparable to human patients may develop more severe CNS lesions. Conversely, it may be postulated that the lesions are not indicative of direct infection of the CNS. The lesions might be mediated through secondary messengers, i.e., cytokines, responding to the systemic viral infection. Studies have demonstrated increased levels of circulating cytokines such as tumor necrosis factor and interleukin-1 in HIV-infected individuals (Lahdevirta et al., 1988; Lepe-Zuniga et al., 1987). Circulating or local cytokine production may be the underlying factor in the pathogensis of neuropathologic lesions without direct infection of the CNS. The inability to demonstrate viral antigen using immunocytochemistry and the lack of multinucleate giant cells leave unresolved the question of whether the virus actually infects CNS tissues in this model. In order to resolve this question, demonstration of viral presence in the CNS is necessary. This might be accomplished by virus isolation, demonstration of intrathecal synthesis of antibodies to viral antigens, or demonstration of HIV sequences using in situ hybridization or polymerase chain reaction techniques. Until virus or viral antigen can be demonstrated in central nervous tissues, the pathogenesis of histologic lesions remains speculative.
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Comparison with other lentivirual infections Relatively few animal models using HIV-l are currently available to the research community. Non-human primates such as chimps (Pan troglodytes) and gibbons (Hylobates lar) have previously been found to be susceptible to persistent infection with HIV-l as evidenced by development of high titer virus-specific antibody responses following infection (Lusso et al., 1988; Alter et al., 1984). However, neither or these species develops clinical signs of infection or pathologic lesions (Watanabe et al., 1991). The endangered status of these species further limits the utilization of these models. Although the modeling of HIV-l infection in these species may offer some utility in vaccine development research, the limitations of these models preclude their use in many areas of HIV investigation. An alternative animal model of HIV-1 is the severe combined immunodeficient @CID) mouse reconstitued with human cells, human fetal liver cells, orhuman bone marrow (Mosier et al., 1988; McCune et al., 1988; Lubin et al., 1991). A SCID mouse reconstituted with human peripheral blood lymphocytes (hu-PBLSCID) has been developed which is susceptible to infection with HIV-l isolates (Mosier et al., 1991). These models may prove useful in addressing questions related to depletion of CD4 + T-lymphocytes in HIV-infected patients. However, the lack of correlation between in vivo pathogenicity and in vitro properties appears to limit the usefulness of these models for investigation of pathogenic potential in vivo (Mosier et al., 1993). In summary, it appears that the HIV-l infected Macaca nemestrina may represent a new animal model for aspects of human HIV infection. Animals inoculated with these strains of HIV-l become persistently infected and display evidence of neuropathologic lesions in the CNS4’. Although virus has not been demonstrated in central nervous tissues, these data indicate that infection is temporally associated with development of lesions. As such, this model may prove useful for future studies in HIV pathogenesis, treatment, vaccine development, and maternal-fetal transmission. These prelimary data suggest that this model may become a useful tool in the continuing efforts to develop effective measures to combat the worldwide AIDS epidemic. Acknowledgements
The authors would like to thank Dr. Clayton Wiley and Dr. Carol Petit0 for their immunochemical examination of tissue sections. We thank Dr. Mark Sumi for his assistance in neuropathologic evaluation of tissues. We would also like to thank Charlene Karr-May for her technical assistance. This work was supported by grants USPHS RR00166 and Comparative Medicine Training Grant RR07019. References Agy, M.B., Frumkin, L.R., Corey, L., Coombs, R.W., Wolinsky, S.M., Koehler, J., Morton, W.R. and Katze, M.G. (1992) Infection of Macaca nemestrina by human immunodeficiency virus type-l. Science 257, 103-6.
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