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1 AIDS Reviews 2004;6:40-53 What Can Natural Infection of African Monkeys with Simian Immunodeficiency Virus Tell us About the Pathogenesis of AIDS? Vanessa M. Hirsch Laboratory of Molecular Microbiology, NIAID, NIH, Rockville, MD, USA Abstract The simian immunodeficiency viruses are a diverse group of viruses that naturally infect a wide range of African primates, including chimpanzees, African green monkeys (AGM) and sooty mangabey monkeys (SM). Although natural infection is widespread in feral populations of AGMs and SMs, this infection does not result in immunodeficiency. However, experimental inoculation of Asian macaque species results in an immunodeficiency syndrome that is remarkably similar in pathogenesis to human AIDS. Thus, SIVsm infection of macaques results in AIDS, and similarly experimental inoculation of pigtailed macaques with at least one SIVagm isolate, SIVlhoest or SIVsun, results in AIDS. The extent of plasma viremia in pathogenic infection is an excellent prognostic indicator of clinical course, with higher viral load being predictive of shorter survival and low viremia being predictive of longterm non-progression. Based upon this paradigm, one would have expected naturally infected animals to exhibit low levels of viremia. In reality, AGMs, SMs, mandrills and chimpanzees infected naturally with their own unique viruses display moderate to high levels of plasma viremia. A significant reduction in CD4+ T-cells in infected versus uninfected SMs suggests that the virus may be cytopathic to some degree. These infected animals still maintain adequate CD4+ T-cells over their entire life in captivity. A distinct characteristic of natural infection is the lack of immunopathology as demonstrated by normal lymph node morphology, lower expression of activation and proliferation markers on CD4+ T-cells, and a generally muted immune response to the virus. Naturally infected SMs and AGMs clearly mount antiviral cellular and humoral immune responses. Therefore, models suggesting immune tolerance to SIV are far too simplistic to explain the lack of disease in these animals. It is probable that a unique balance between T-cell renewal and proliferation and loss through activationinduced apoptosis, and virus-induced cell death has been achieved in SMs and AGMs. The study of the dynamics of T-cell production, proliferation and cell death in asymptomatic natural infection should, therefore, yield insights into the pathogenesis of AIDS. (AIDS Rev 2004;6:40-53) Key words SIV. Pathogenesis. Animal models. Long-term non-progressors. 40 a wide range of African primates, and are the source Genetic relationships No of part SIVs and of this HIV publication of the human may immunodeficiency be viruses (HIV-1 and HIV-2). Many of the SIV strains can be categorized The simian immunodeficiency viruses (SIV) are a genetically based upon their natural host species, as genetically diverse group of viruses that naturally infect recently reviewed 1,2. Basically, primate lentiviruses have been detected in most if not all of the African Correspondence to: Vanessa M. Hirsch monkeys of the genus Ceropithecus, the closely related African green monkeys (genus Chlorocebus), Laboratory of Molecular Microbiology NIAID, NIH, Twinbrook II Facility mangabeys (Cercocebus), colobus monkeys (Colobus), and within two subspecies of the chimpanzees Parklawn Drive Rockville, MD 20852, Permanyer USA Publications (genus Pan) of the great apes (Table 1). But interestingly, infection of Asian monkeys such as vhirsch@niaid.nih.gov macaques

2 Vanessa M. Hirsch: Natural Infection of African Monkeys Table 1. Characterization of Simian Immunodeficiency Viruses and Their Natural Host Species Natural Host Species Strain Members Common Name Scientific Name Complete Sequences Available SIVsm SIVmac, SIVmne, SIVstm Sooty mangabey Cercocebus torquatus atys HIV-2 SIVrcm Red-capped mangabey Cercocebus torquatus torquatus SIVagm SIVagm/Tan Tantalus monkey Chlorocebus tantalus SIVagm/Ver Vervet monkey C. pygerythrus SIVagm/Gri Grivet monkey C. aethiops SIVagm/Sab Sabaeus monkey C. sabaeus SIVlhoest SIVlhoest L Hoest monkey Ceropithecus l hoesti l hoesti SIVsun Suntailed monkey C. l hoesti solatus SIVmnd-1 Mandrill Mandrillus sphinx SIVsyk Sykes monkeys Cercopithecus albogularis SIVcol Black and white colobus Colobus guereza SIVcpz SIVcpz/Ptt Chimpanzee Pan troglodytes troglodytes SIVcpz/Pts Pan troglodytes schweinfurthi SIVmnd/drl SIVmnd-2 Mandrill Mandrillus sphinx SIV drl Drill Mandrillus leucophaeus SIVgsn Greater spot-nosed guenon Cercopithecus nictitans Partial Sequences Available SIVtal Talapoin Cercopithecus talapoin SIVwrc Western red colobus Colobus polykomos SIVolc Olive colobus Pilocolobus badius SIVdeb Debrazza s monkey Cercopithecus nictitans SIVmon Mona monkey Cercopithecus mona SIVasc Redtailed monkey Cercopithecus ascanius schmidti (genus Macaca) and the Asian great apes, orangutans, has not been detected in the wild. This restriction of primate lentiviruses to African monkeys suggests that this is an ancient virus that has coevolved with its primate host. The ancestor of current day SIVs may be as ancient as the time of phylogenetic divergence and geographical separation of African and Asian monkeys. In contrast, HIV-1 and HIV-2 infections of humans represent relatively recent introductions into human populations by cross-species transmission 4. As detailed in table 1, there are at least six distinct lineages of primate lentiviruses: 1) SIVsm from sooty mangabeys and including HIV-2 5 ; 2) SIVagm from the four different species of African green monkeys (vervet, grivet, tantalus and sabaeus 6-15 ); 3) SIVsyk from Sykes monkeys 16 ; 4) SIVcpz from two species of chimpanzees ; 5) SIVlhoest and SIVsun from the related L Hoest and suntailed monkeys 20,21, and 6) SIVcol from guereza colobus monkeys 22. Each of the various lineages is approximately equidistant from any other lineage, sharing 40 to 50% identity in the most conserved Gag and Pol proteins 1,2. The phylogenetic relationship between many of these various SIVs is shown in figure 1, with distinct species-specific lineages, and HIV-1 intermingled within the SIVcpz group and HIV-2 within the SIVsm group. This tree of a portion of Gag shows the clustering of SIVmnd-type 1 with viruses of the L Hoest lineage (SIVlhoest and SIVsun) and SIVmnd-type 2 sequences clustering with SIVdrl 23. Mandrills are unique in the circulation of two distinct viruses within feral populations 23,24. Recently, full length sequences of novel SIV strains have been characterized from SIVrcm from red- Permanyer Publications

3 AIDS Reviews 2004;6:40-53 SIVsyk173 SIVmac251 SIVmac239 SIVstm SIVsm9 HIV-2A/ALI HIV-2A/ST HIV-1B/D205 HIV-2B/EHO HIV-2G/ABT96 SIVagmTAN1 SIVagmVER9063 SIVagmVER155 SIVagmVER/TYO SIVagmVER3 SIVagmSAB1C SIVagmGRI677 SIVrcmNG411 SIVrcmNG409 HIV-1A_UG.U455 HIV-1 F_BE.VI85 HIV-1 B_FR.HXB2 HIV-1 D_ZR.84ZR HIV-1 G_SE.SE61 HIV-1 C_ET.ETH2 HIV-1 N_CM.YBF3 SIVcpzUS HIV-1 O_CM.MVP5180 HIV-1 O_BE.ANT70 SIVcpzANT SIVmnd2-S46 SIVmnd SIVmnd2-S6 SIVmnd2-9 SIVmnd2-13 SIVmnd2-14 SIVmnd2-7 SIVmnd2-99CM31 SIVmnd2-98CM1 SIVmnd2-98CM16 SIVmnd2-US.BK SIVmnd2-S109 SIVmnd2-302 SIVmnd2-99CM5 SIVmnd2-PG13 SIVmnd2-99CM33 SIVmnd2-99CM35 SIVdrl007 SIVdrl006 SIVmnd2-99CM32 SIVdrl1FAO SIVdrl96602 SIVdrl1207 SIVmnd1-GB1 SIVsun SIVlhoest7 SIVlhoest524 SIVlhoest485 SIVlhoest447 SIVcolCGU1 FIVPETLM FIVUSCAPP FIVUSIL24 FIVJPTM2 FIVKZOMA PLV14CG HIV-2 and SIV-SM SIV-AGM SIV-RCM FIV-Wild cat.10 HIV-1 and SIV-CPZ SIV-MND-2 SIV-DRL SIV-LHOEST FIV-Domestic cat 42 Figure 1. The phylogenetic Permanyer relationship between of the various Publications SIV and HIV strains. Un-rooted phylogenetic 2010 tree (maximum likelihood with site-specific substitution rates) of a conserved fragment of the pol gene including many of the available SIVmnd and SIVdrl sequences. The alignment was 397 nucleotides after gap-stripping.

4 Vanessa M. Hirsch: Natural Infection of African Monkeys capped mangabeys 25, SIVgsn from greater spot-nosed monkeys 26, SIVdrl and SIVmnd-2 from drill and mandrills 23. These sequences appear to be recombinant and thus cannot be clearly characterized as distinct lineages. Partial sequences have also become available from two different species of colobus monkeys 28, SIVwrc and SIVolc from western red colobus and olive colobus monkeys, which interestingly do not cluster phylogenetically with SIVcol 21. Other recent sequences reported include SIVmon from mona monkeys 29, SIVdeb from DeBrazza monkeys 3, SIVtal from talapoins 30, and SIV from red-tailed monkeys 31. The initial view of the primate lentiviruses suggested that virus lineages segregated with an individual host species, consistent with co-evolution during the speciation and migration of the different primate species throughout Africa. However, as more and more SIV strains are isolated from different species of African primates, the picture has become increasingly more complex. There is clear evidence of cross-species transmission between monkey species 32,33 as well as recombination 18, Thus SIVagm has been isolated from patas monkeys and yellow baboons that were presumably infected by virus from sympatric species of African green monkeys 32,33. Many of the SIV strains such as SIVcpz, SIVdrl, SIVmnd-2, SIVagm/Sab and SIVrcm appear to be recombinant, and often the parental strains are difficult to define due to genetic divergence from representative strains. SIVdrl and SIVmnd-2 are the clearest recombinants between SIVrcm and SIVmnd-1, and share a common breakpoint suggesting a common origin and another example of cross-species transmission 27. SIVcpz also appears to be a recombinant between SIVrcm-like virus and SIVgsn-like virus 19. However, the genetic divergence of SIVcpz from either SIVrcm or SIVgsn makes the actual parental strains difficult to define precisely. Presumably there has been a long history of cross-species transmission events and recombination. The origins and divergence of the various SIVs from a common ancestor cannot be precisely defined, but it is evident from the species specificity of many of these strains that these viruses are ancient and have coevolved with their host species over long periods of time. long period of adaptation from initial virulence to select for disease resistance over time, or SIV may have always resulted in asymptomatic infection. Neither scenario can be ruled out. Clearly these viruses are not inherently apathogenic, since experimental or accidental inoculation of Asian macaques can result in an AIDSlike disease 36,37. This discrepancy in disease outcome was first observed with SIVsm infection of macaques, and subsequently with one SIVagm 38 isolate in pigtailed macaques, and SIVlhoest and SIVsun in pigtailed macaques 20,21. As reviewed previously 36,37,39 and as described below, SIV infection of macaques is remarkably similar in pathogenesis to human AIDS. SIVsm and SIVmac in rhesus and pigtailed macaques A variety of viruses derived from SIVsm have been studied in macaques (Table 2). These vary in pathogenicity for macaques from highly attenuated (SIVmac1A11) to highly pathogenic strains such as SIVmac239, SIVmac251 and SIVsmE Even within macaques inoculated with a common SIV strain, the disease course varies considerably from rare, longterm non-progression to rapid progression in a period of less than six months from inoculation 36,37. Similar to HIV infection in humans 40, the levels at which viremia plateaus following primary viremia is predictive of disease outcome in SIV-infection 41,42. Thus, macaques with low levels of viremia (<1.000 copies/ml plasma) remain disease free longer than macaques with high levels of persistent viremia (10 6 /ml). The pathogenesis of SIVsm/mac infection in macaques is remarkably similar to human AIDS, with the exception of a significantly more rapid disease course of 1-2 years median survival. Both show a period of intense viremia during primary infection that persists, but becomes modulated somewhat with the development of a CTL response and neutralizing antibody, followed by a variable asymptomatic period. This period is characterized by persistent viral replication 41,42,46,47, lymphadenopathy 47,48, strong humoral and cellular immune responses, and a slow but progressive decline in numbers of circulating CD4+ T-lymphocytes. CTL 49,50 and neutraliz- ing antibody escape mutants develop throughout Pathogenic infection of Asian macaques pathogenic infection and are associated with increased monkeys levels of plasma viremia and more rapid eventual progression to AIDS. Opportunistic infections and clinical One of the intriguing features of SIVs is that they result AIDS generally are observed when peripheral CD4 in asymptomatic infection Permanyer in their natural African Publications host counts drop to less than cells/µl, although many species (reviewed in ). This could be the result of a macaques that die very rapidly may show normal CD4 43

5 AIDS Reviews 2004;6:40-53 Table 2. Experimental pathogenicity of simian immunodeficiency viruses SIV subgroup Common Uncloned virus stocks Molecular cloned virus classification designation Name Disease* Clones Disease Sooty Mangabey & Macaque Cercocebus atys SIVsm/SIVsmm SIVsm/F236 AIDS, slow (R,P) SIVsmH3, smh4 AIDS/slow (R,P) (sooty mangabey) SIVsm/B670 AIDS, rapid (R) n.a. n.a. SIVsmm9 AIDS, rapid (R,P) n.a. n.a. SIVsm/E660 AIDS, intermed.(p) n.a. n.a. SIVsm/E543 AIDS, rapid (P) SIVsmE543-3 AIDS (RP) SIVsmm/PBjbcl3 Acutely lethal (P) PBj4.41 Acutely lethal (P) PBj6.6 Acutely lethal (P) SIVmac/SIVmm SIVmac/251 AIDS, rapid (R) SIVmac251 None (R) SIVmac/BK28 Minimal (R) SIVmac/1A11 None (R) SIVmac239 AIDS, rapid (R) SIVmac239 AIDS, rapid (R) SIVmne SIVmne AIDS, intermed. (P,R) SIVmne/E11S AIDS, slow (R,P) SIVstm SIVstm AIDS, rapid (S,P) SIVstm/37.16 AIDS, slow (P) African green monkey Cercopitthecus pygerythrus SIVagm/ver SIVagm155 Rare (R,P,A) SIVagm/155-4 None (R,P,A) (vervet) SIVagm/TYO None (A,C) SIVagm/TYO-1 None (A,C) SIVagm3 None (A,C) SIVagm3mc None (A,C) SIVagm/385 None (R) n.a. n.a. SIVagm/266 None (R) n.a. n.a. SIVagm/90 AIDS (PT) SIVagm9063 AIDS (P), None (A,R) Cercopithecus aethiops SIVagm/gri SIVagm/gri None (A,R) SIVagm/gri-1 n.t. (grivet) Cercopithecus tantalus SIVagm/tan SIVagm/tan n.t. SIVagm/tan-1 n.t. (tantalus) Cercopithecus sabaeus SIVagm/sab SIVsab-1,2 None (A,R) SIVsab-1 n.t. (sabaeus) *n.t. = not tested; n.a. = not applicable; P = pigtailed macaque; R = rhesus macaque; C = cynomologus macaque; rapid is defined as 25% death by 3 months, and two thirds by 1 year post inoculation; slow is defined as no deaths before 1 year; intermediate indicates that no deaths by 3 months but one third death by 1 year; none indicates no evidence for AIDS in inoculated animals; minimal indicates rare deaths from AIDS occurring > 1 year post inoculation 44 T-cells at the time of death 42. Animals die from a variety fected patients suggest that CD4+ T-cell virus-induced of opportunistic infections such as rhesus cytomegalovirus (CMV), Pneumocystis pneumonia, and Mycobac- AIDS. Rather, HIV-1 pathogenesis appears to be the cell death cannot alone explain the pathogenesis of terium avium infection 55,56. Kaposi s sarcoma is not result of a complex interplay between the virus and the observed in the SIV/macaque models. Similar to HIV, immune system that involves early destruction of SIV also can result in neuro-aids due to meningoencephalitis, associated with the evolution of neurotropic crease in memory T-cell turnover, thymic dysfunction, memory T-cells by virus, followed by an overall in- variants of SIV 57. and limitations in peripheral T-cell regeneration. Spontaneous apoptosis of T-cells is increased, apparently Complex mechanisms No for part AIDS? of this publication through bystander may mechanisms be since both CD4 and CD8 T-cells are affected. This increased apoptosis is The correlation between plasma reproduced viremia and rapidity or photocopying thought result from increased T-cell activation in of disease progression in pathogenic SIV and HIV-infections suggests that much of the CD4 depletion creased levels of cellular activation markers (such as SIV-infected macaques and human AIDS patients. In- that occurs in AIDS is the direct result of virus-induced HLA-DR) in both CD4 and CD8 T-cells are observed. cell death. However, although extensive virus replication occurs throughout the disease course, of the immune the publisher cell proliferation, as measured by proliferation markers In addition there is evidence of increased levels of T- system deteriorates very gradually, suggesting that such as Ki-67, and increased incorporation of BrdU other mechanisms Permanyer are important in the development Publications of into T-cell DNA. Figure 2 schematically 2010 shows the AIDS. As recently reviewed 58,59, studies with HIV-in- complex interplay between T-cell renewal from the

6 Vanessa M. Hirsch: Natural Infection of African Monkeys RENEWAL TRAFFICKING Bone marrow Lung Spleen Naïve CD4+ T cells Blood Thymus Gut associated Lymphoid tissues ANTIGEN DRIVEN PROLIFERATION AND APOPTOSIS Antigen presenting cells cytokines GC ACTIVATION Lymph Nodes Paracortex Proliferation Naïve CD4+ T cells Activated T cells Resting Memory T cells Programmed cell death CD4 and CD8 INFECTION Infected without Cell death the prior written CD4+ T cells permission Infected Macrophages Permanyer Publications 2010 Figure 2. Overview of the potential role of T-cell renewal, activation-induced cell death and trafficking of T-cells in pathogenic and natural lentiviral infection. Potential areas of difference between natural and pathogenic infection are marked with a star. 45

7 AIDS Reviews 2004;6: thymus and bone marrow, trafficking to tissues, and antigen-driven proliferation and apoptosis. Other pathogenic SIV/macaque models SIVagm in pigtailed (PT) macaques. Only one isolate and clone of SIVagm that induces disease in PT macaques has been described 38. SIVagm90 was originally isolated from a naturally infected African green monkey (vervet) and induced AIDS in a PT macaque. SIVagm9063 isolated from this PT macaque is clearly pathogenic in PT macaques, but produces no disease in rhesus macaques or AGMs (to be discussed later). The pathogenesis is similar to SIVsm/ mac in rhesus macaques, with some minor differences; a slower rate of disease progression and an earlier peak in primary viremia. However, SIVagm is still highly pathogenic in PT macaques, resulting in progressive CD4 depletion and rapid progression to AIDS in macaques 38. Levels of primary viremia are similar to those observed in SIVsm/mac infection, but peak earlier (one week versus two weeks for SIVsm/mac) and there is a subsequent decline to a characteristic plateau level for each individual animal (Hirsch, unpublished data). As with SIVsm/mac infection, the plateau levels of viremia correlate with the rate of disease progression. SIVagm isolates may not be uniformly pathogenic in PT macaques, since very few have been evaluated (Table 2). Most of the isolates evaluated were from the vervet subspecies (SIVagm/Ver). The majority of studies of SIVagm in macaques have utilized rhesus or cynomolgus macaques rather than PT macaques. In addition, earlier studies with the pathogenic SIVagm9063 isolate demonstrated that, although this isolate was pathogenic in PT macaques, it did not replicate well in highly related rhesus macaque monkeys, as evidenced by difficulty in isolating virus from the blood following infection and unapparent plasma antigenemia during primary infection 38. Therefore, many of the studies in which SIVagm isolates were not apparently pathogenic may be due primarily to the use of the most susceptible host species. In a recent unpublished study, my laboratory evaluated the original SIVagm90 isolated directly from tissues of the naturally infected vervet, AGM90 and another natural isolate, SIVagm155. These studies confirmed that SIVagm90 was pathogenic in macaques without any apparent adaptation, but suggested that the macaquepassaged isolate was slightly more virulent (unpublished data). Although SIVagm155 resulted in high primary viremia, macaques infected with this isolate controlled viremia and have remained asymptomatic for over one year. Thus, there is clearly considerable variability in the pathogenic potential of SIVagm isolates in macaques and the mechanisms underlying this difference are not clear. SIVlhoest and SIVsun in macaques. Similar to SIVagm, SIVlhoest and SIVsun infection of PT macaques results in an immunodeficiency syndrome, characterized by progressive declines in CD4+ T-cell numbers in the blood and tissues and the development of opportunistic infections ( 20,21 and unpublished observations). Macaques develop lymphadenopathy concurrent with a transient increase in SIV-expressing cells in lymph nodes, and trapping of virus on germinal centers is observed. This virus apparently required no adaptation to macaques to become pathogenic. SIVlhoest- and SIVsun-infected macaques appeared to survive longer than SIVsm/mac-infected macaques, and progression was associated with low viremia during the chronic phase of infection. As with other experimental macaques models, animals die due to opportunistic infections ( 20 and unpublished) so the pathogenesis of all these viruses in macaques appears very similar to each other and to human AIDS. Natural infection of AGM and SM The lack of virulence of SIV isolates for their particular natural host species is intriguing. African monkeys, such as SMs infected with SIVsm or AGMs infected with SIVagm, exhibit no evidence of immunodeficiency However, experimental infection of Asian species of monkeys (Macaca spp.) with SIVsm, or the highly related SIVmac strain 36,37, or SIVagm 38, can induce an AIDS-like syndrome. SIVlhoest isolated from L Hoest monkeys also appears to induce AIDS in macaques, while apparently resulting in asymptomatic infection in its natural host 20. The comparative study of the pathogenesis of such viruses in their natural host, and a disease-susceptible host such as macaques, thus may shed light on the pathogenesis of AIDS in humans. This review will summarize briefly what we currently understand about natural infection. More detail can be found in specific reviews of AGM 34 and SM infection 35 of their respective natural hosts. The paradigm that pathogenesis is associated with the extent of viral replication does not appear to be maintained in examining the natural models of SIV infection. Studies of viral load in naturally infected SM monkeys demonstrated active ongoing viral replication as Permanyer Publications 2010

8 Vanessa M. Hirsch: Natural Infection of African Monkeys measured by plasma viral RNA assays 60. Since the viral load in asymptomatic mangabeys is comparable to that observed in macaques inoculated experimentally with pathogenic SIV 34-37, it appears unlikely that strict containment of viremia is a likely explanation for the lack of pathogenicity of SIVsm in its natural host species. Natural infection is generally asymptomatic A relatively small number of naturally infected African primates have been studied in terms of virologic and immunologic parameters. These include AGMs of the vervet, sabaeus and tantalus species, SMs, mandrills and chimpanzees. Infection of these species results in asymptomatic infection with few notable exceptions. While it is difficult to assess the effect of SIV infection in wild populations, a large number of AGMs and SMs have been studied in captivity over long periods of time 34,35. The conclusion is that SIV infection of natural host species is generally asymptomatic. CD4 depletion was observed in one naturally infected mandrill, suggesting that in some circumstances these viruses may produce AIDS in their natural host, perhaps after long incubation periods 61. AIDS has also been observed in at least one chimpanzee inoculated with HIV-1 and was associated with an increase in viremia terminally, suggesting the evolution of a pathogenic variant of HIV While HIV-1 is only a close relative of the chimpanzee virus, SIVcpz, this study suggests that these African primates are not immune to the pathogenic effects of primate lentiviruses under specific circumstances. It is not clear whether disease development is associated with the evolution of a more pathogenic variant or perturbation of the normal balance between virus and host. observed among naturally infected AGMs 66, with more consistently high viremia observed in SMs and mandrills 60,68. These comparisons use different assays and thus direct comparisons are not entirely valid. Therefore, it is possible that there are threshold differences between natural infection and infection of macaques, and that boosting of virus load by an order of magnitude in AGMs or SMs might result in disease expression. Genetic evolution of SIV in both AGMs and SMs also suggests ongoing, persistent viral replication 35,60,71. The degree of in vivo evolution of virus is affected as much by selective pressures, such as immune responses, as it is by the extent of viral replication. Only under situations of highly effective antiretroviral therapy (<50 copies/ml of plasma) does viral evolution appear to cease. Therefore, viral evolution in these animals simply confirms active and persistent viral replication, but cannot be used to assess viral load. Regardless, natural infection of SMs, AGMs and mandrills results in moderate to high levels of viremia, frequently in the range associated with disease progression in macaques infected with SIV. Thus, natural models of SIV are not analogous, in virologic terms, to long-term non-progressors of HIV infection. Target cells and distribution of SIV in natural infection SIVsm and SIVagm both utilize CCR5 and CD4 as receptors and thus preferentially infect T-cells and macrophages. Interestingly, CD4+ T-cells of AGMs co-express CD8 antigen 74 ; the significance of this in terms of pathogenesis in not known. AGMs also show a high degree of polymorphisms in CCR5 genes, which could influence the extent of viral replication 75,76. Similar to HIV in humans 77, the target cells of SIV in macaques are CD4+ CCR5+ memory T-cells that are Moderate to high viremia in natural predominantly found in mucosal sites such as the infection gastrointestinal tract, lung and urogenital tract 78,79. In fact, marked CD4 depletion occurs early in mucosal The initial assumption regarding No part the lack of of this disease publication sites in SIV-infected may macaques be 77, presumably through in natural host species such as AGMs was that it resulted from strict containment virus-induced cell death. The target cells and distribution of virus natural infection have been studied in a reproduced of viral replication, or photocopying analogous to what is observed with long-term nonprogressors of HIV-infection. However, this is clearly CCR5 was observed CD4+ T-cells of SIV-infected limited fashion. Interestingly, decreased expression of not the case. Naturally infected AGMs 63-67, SMs 60,68 versus uninfected SMs, suggesting that downregulation of CCR5 or loss of CCR5+ T-cells may play a role and mandrills 69,70 appear to have significant, ongoing viral replication similar to levels during the of asymptomatic phase of pathogenic infection in macaques and Lymph nodes of SIV-infected SMs contain moderate the publisher in limiting target cells for the virus in these animals 79. humans. Thus, plasma Permanyer viral RNA levels range from Publications 10 3 numbers of SIV-expressing cells SIV appears to infect CD4+ T-cells and macrophages in naturally to 10 7 copies per ml of plasma. A wider range was in- 47

9 AIDS Reviews 2004;6: fected AGMs, and is distributed in lymphoid tissues such as the spleen, lymph nodes, and thymus, as well as the gastrointestinal tract. A large number of pulmonary macrophages 38 were observed in one AGM (Agm90). This animal exhibited moderate viremia, leading to speculation that much of the virus replication in this animal may have been occurring in macrophages rather than T-cells. Such preferential targeting of macrophages might provide an explanation for the lack of CD4 T-cell depletion in natural infection, but would require confirmation in additional animals. Virus was not detected in the brain of two AGMs of the vervet species 65, and further unpublished studies of viral RNA in cerebral spinal fluid (CSF) of naturally and experimentally infected vervets showed no evidence of infection in this compartment. However, one study observed high levels of virus in the brain and CSF of naturally infected AGMs of the sabaeus species, in the absence of neurologic disease or neuropathology 63. Comparable studies of the brain and CSF have not been performed in naturally infected SMs. Thus, the distribution of SIV in natural infection appears to be similar to that observed in pathogenic models in macaques. Complex mechanisms for asymptomatic infection? Clearly the lack of disease in naturally infected monkeys cannot be explained by strict containment of viremia. This leaves factors such as the balance between T-cell regeneration from the thymus and from homeostatic proliferation in the peripheral lymphoid tissues, the extent of virus-induced T-cell killing, the amount of activation-induced apoptosis, and cell-mediated immune lysis (Fig. 2). Multiple scenarios can be postulated that might explain the lack of disease in naturally infected hosts in the face of persistent viremia: 1) the degree of cytopathic effect of the virus on target CD4+ T-cells; 2) the actual target cells infected, i.e. T-cells versus macrophages; 3) thymic function and the efficiency of T-cell regeneration from the thymus; 4) homeostatic proliferation of T-cells in the peripheral lymphoid tissues, and 5) the extent of activation and activation-induced bystander apoptosis. These various pathways are shown schematically in figure 2. Moderate to high viremia in these animals suggests that other immune mechanisms may play a role in protecting these animals from AIDS. It also suggests that our understanding of the pathogenesis of AIDS is incomplete. Clearly AIDS is not entirely the result of virusinduced CD4 T-cell death, but rather may result from the perturbation of the balance between thymic output and regeneration of T-cells, and activation and virusinduced cell death. Is SIV cytopathic in natural infection? One potential and attractive hypothesis to explain the lack of disease in natural infection is the lack of cytopathic effects or virus-induced cell killing in these hosts. This is a difficult issue to address in vitro, although SIVsm is reported to be less cytopathic in mangabey cells than in rhesus cells 81. There are two lines of evidence that suggest that SIVsm may be cytopathic for CD4+ T-cells in vivo. First, comparison of CD4+ T-cells in the blood of infected versus uninfected SMs revealed a significantly lower CD4+ T-cell numbers in the infected cohort 35. However, the levels were still within what would be considered a normal range and certainly not consistent with AIDS. Preliminary studies of the dynamics of viral replication in SMs subjected to short term antiretroviral therapy (PMPA) revealed a rapid decline in viral load following treatment, suggesting that the half-life of infected cells is short 35,91. However, this is only an indirect measurement of the half-life of infected CD4 T-cells in such animals, and clearly further studies are required to evaluate the direct effects of virus on CD4 cells in natural hosts. In summary, these studies suggest that SIV is cytopathic to some degree in SMs, but all of the measures are relatively indirect, and there may be quantitative differences from the degree of cytopathology in pathogenic infection. Reduced immune activation in natural infection The most obvious difference between pathogenic and natural infection is the lack of intense hyper-immune activation in natural infection. As shown in this low-power micrograph of peripheral lymph nodes from experimentally infected PT macaques and AGMs, SIV-infected macaques develop lymphadenopathy with paracortical and follicular hyperplasia early (Fig. 3). Progression to AIDS and CD4 depletion is associated with depletion in paracortical areas and involution of germinal centers in lymph nodes and other lymphoid organs. Infection is also associated with an increase in the number of T-cells expressing activation and proliferation markers. In contrast, SIV-infected AGMs and SMs display normal lymph node morphology without evidence of either hyperplasia Permanyer Publications 2010

10 Vanessa M. Hirsch: Natural Infection of African Monkeys A B Figure 3. Comparison of hematoxylin eosin stained sections of peripheral lymph nodes from experimentally SIVagm-infected PT macaque (panel A) and SIVagm-infected AGM (panel B) showing the dramatic lymphadenopathy in the macaque and normal lymph node morphology in the AGM. These lymph nodes were biopsied at 8 weeks after inoculation with SIV. or depletion. Activation and proliferation markers are generally not up-regulated in natural infection 68,82. The major difference in lymphoid pathology between macaques and natural hosts suggests that much of the pathogenesis of SIV is an indirect result of hyper-immune activation driving excessive activation-induced apoptosis. This, in conjunction with impaired regeneration of T-cells from the thymus and the steady loss of CD4 T-cells by direct virus-induced cytopathic effects, may ultimately lead to the collapse of the immune system in AIDS 58,59. The dynamics of T-cells can be evaluated by assaying telomere length as a surrogate of T-cell proliferation, measurements of proliferation using Ki67 expression, and direct labeling of dividing cells using 5-bromodeoxyuridine (BrdU). Definitive studies of SMs and AGMs have not been performed at the present time. However, SIV infection of macaques and HIV infection of humans clearly increases the fraction of proliferating T-cells and T-cell turnover Immune mechanisms in natural infection With the exception of macaques which progress rapidly to AIDS, macaques infected with SIVsm/mac develop robust neutralizing and cellular immune responses and exhibit continual immune escape 50,53,54. The immune responses in natural infection are difficult to compare with those of macaques, particularly with respect to cellular assays. However, the general consensus is that de novo immune responses in natural infections are muted compared to those in pathogenic models 88. This has lead to the suggestion that the natural host species develop tolerance to their virus or to specific antigens or epitopes 90,91. This explanation is too simplistic since it is evident that these animals do indeed mount immune responses. However, their immune responses may differ qualitatively and quantitatively from animals with pathogenic infection. SIV-infected AGMs and SMs develop antibody responses to viral proteins but, unlike ma- Permanyer Publications

11 AIDS Reviews 2004;6: caques, the predominant responses are directed to the model for natural SIVsm infection, since this virus envelope proteins rather than gag proteins 90. While it is has been adapted by animal passage to replicate unlikely that Gag-specific antibodies per se are harmful, efficiently in rhesus macaques. Indeed, comparisons the lack of such antibody responses in naturally infected of SIVmac239-infected and naturally infected SMs species suggests some difference in the host immune revealed lower plateau levels of virus and more robust CTL responses 92. Our laboratory has focused response that may explain the lack of disease in these animals. Cytotoxic T-cell responses have been characterized less extensively and have focused on SMs. Clearly SIVagm90, initially utilizing a macaque-passaged on the use of a pathogenic SIVagm/Ver isolate, SIV-infected SMs develop CTL responses 88 and CTL variant, SIVagm9063 (Hirsch.) Our initial comparative studies of AGMs and PT macaques utilized escape can also be documented 89, suggesting that the CTL responses are functional to some degree in controlling virus replication. It is difficult to compare the strength typically appear to be of the sabaeus species rather AGMs imported from the Barbados, which pheno- of CTL responses in macaques and SMs; however, CTL than vervet, the source of the virus isolate 38. Lack of responses are generally more robust in macaques than antigenemia in these AGMs during primary viremia in SMs. Studies to dissect the role of CD8 T-cells in controlling viral replication in SMs and AGMs have not been caques inoculated in parallel. These data suggested contrasted with significant antigenemia in PT ma- performed. that control of viremia might be a major determinant of the lack of disease in AGMs as compared with Thymic and bone marrow function macaques 38. However, recent unpublished studies comparing plasma viremia in AGMs inoculated with Pathogenic SIV and HIV infections may perturb normal T-cell regeneration, partly through an effect on T- SIVagm9063 demonstrated more efficient replication the primary SIVagm90 and the macaque-passaged cell production from the thymus and bone marrow (see of SIVagm90 isolate, suggesting that SIVagm9063 renewal portion of Fig. 2). Thymic output has not been has been adapted to macaques (unpublished data) studied extensively in either pathogenic or nonpathogenic SIV models that can be investigated indirectly ral infection of AGMs (similar to SIVmac239). The and thus might not represent a valid model for natu- by assaying for T-cell receptor excisional circles exact species of AGM also appears to be critical for (TREC). TRECs are produced during T-cell maturation such studies, since comparisons of SIVagm90 (a in the thymus and thus are an indirect measure of recent thymic emigrants to the blood. TREC levels have vervets (C. pygerythrus) revealed higher primary and vervet isolate) in Barbados greens (C. sabaeus) and been observed to decrease in both CD4 and CD8 set-point viremia in the vervets than in the mismatched sabaeus monkeys. Primary viremia was populations by 16 to 30 weeks following pathogenic SIVsm/mac infection 85, suggesting that thymic function similar to that observed in PT macaques inoculated was impaired in pathogenic SIV infection. In many instances in this study, declining TREC levels correlated levels were higher in those PT macaques that pro- with the same virus, although the plateau viral RNA with an increased level of T-cell proliferation as measured by Ki67 expression 85. However, a comparative Inoculation of sabaeus monkeys with SIVagm/Sab gressed to AIDS than any of the healthy vervets. study of TREC levels in naturally infected SMs and showed high primary viremia (10 6 to 10 8 /ml of plasma) experimentally infected macaques showed only a and significant control of virus following primary viremia to levels comparable to those observed in natu- moderate decrease in both 82. Thus, the role of thymic function in preserving CD4 No subsets part during of natural this infection is still unclear. been observed in mandrills inoculated with a primary publication rally infected sabaeus may monkeys. be A similar pattern has SIVmnd-type 1 isolate 92. Peripheral CD4+ T-cell numbers of the naturally infected SMs, AGMs and man- Experimental infection in natural hosts drills 70,92 were maintained, with the exception of a Experimental models of natural and pathogenic transient decrease during primary infection. The infection allow a more direct comparison immune down-modulation of viremia following primary viremia responses and viral load parameters and of distribution the publisher suggests a relatively efficient immune response controlling viremia. However, studies to deplete immune of virus. To be valid, these studies must utilize viruses that closely Permanyer model natural infection. Thus Publications effector cells such as CD T-cells have not been SIVmac239 infection of SMs may not be a legitimate performed.

12 Vanessa M. Hirsch: Natural Infection of African Monkeys Summary It is still unclear why naturally infected African monkeys remain healthy. One common and conclusive feature of natural infection is persistence of moderate to high levels of viremia and viral replication in naturally infected monkeys. While it is not clear that the levels of viremia are equivalent to the range observed in pathogenic infection, certainly many naturally infected SMs and AGMs exhibit viremia in the range normally associated with disease progression in pathogenic models (i.e /ml). Therefore, the lack of disease in naturally infected monkeys cannot be explained on the basis of effective control of viral replication. However, the range of virus load, while overlapping that in macaques, is clearly lower. Thus, the extremely high viremia observed in macaques that progress rapidly to AIDS following early failure of immune responses 42 has not been observed in either SMs or AGMs Studies of CTL responses in SMs, and experimental infection of mandrills and AGMs, suggest that there is clearly some level of immune control of viremia as shown by a sharp reduction in viremia following primary infection. Another consistent feature of natural infection is the lower level of abnormal immune activation as assessed by the lack of lymphadenopathy, and low levels of activation and proliferation markers of T-cell subsets. Thus, the possible mechanisms underlying the lack of disease include lack of cytopathology for CD4 T-cells, lack of hyper-immune activation-induced apoptosis, and preservation of thymic and bone marrow function. Possibly these naturally infected animals have achieved a better balance between regeneration of T-cells and loss through virus-induced and activation-induced cell death. However, clearly the natural models tell us that we have still much to learn about the actual pathogenesis of AIDS. It is obvious that simplistic models which overemphasize the importance of one specific actor such as virus-induced cell death, or a negative impact of immune responses, are not adequate to explain either the immunodeficiency induced by HIV in humans or the lack of disease in naturally infected nonhuman lentivirus models. Acknowledgments The author thanks Brigitte Beer and Joern Schmitz for their contributions to unpublished data included in this manuscript. nonhuman primate lentivirus family. J Virol 2001;75: Souquiere S, Bibollet-Ruche F, Robertson DL, et al. Wild Mandrillus sphinx are carriers of two types of lentivirus. J Virol 2001;75: Permanyer Publications Peeters M, Courgnaud V, Abela B. Genetic diversity of lentiviruses 24. Hu J, Switzer W, Foley B, et al. Characterization and comparison of in nonhuman lentiviruses. 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