Plateau Levels of Viremia Correlate with the Degre
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病菌学杂志 2005年第8期
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
Bioqual, Inc., Rockville, Maryland
ABSTRACT
Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques (Macaca nemestrina) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4+-T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+-T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4+-T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (P values of <0.01). Considering all the macaques, the degree of CD4+-T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.
INTRODUCTION
The simian immunodeficiency viruses (SIV) are a diverse group of viruses that naturally infect a wide range of African primates (2, 21, 43, 49). At the present time, SIV from at least seven African monkey species have been identified and molecularly characterized. The genetic relationships among many of these SIV strains have been reviewed previously (2, 43, 49-51). Full-length sequences have currently been characterized from sooty mangabey monkeys (Cercocebus atys, SIVsm), mandrills and drills (Mandrillus sphinx and Mandrillus leucophaeus, SIVmnd and SIVdrl) (55, 58, 59), Sykes monkeys (Cercopithecus albogularis mitis, SIVsyk) (23), African green monkeys (AGM), (Chlorocebus sp., SIVagm), chimpanzees (Pan troglodytes, SIVcpz) (4, 16, 47, 48), redcapped mangabeys (Cercocebus torquatus, SIVrcm), colobus monkeys (Colobus guerza, SIVcol), and a number of African guenons, including greater spot-nosed guenons (Cercopithecus nictitans, SIVgsn), DeBrazza monkeys (Cercopithecus neglectus, SIVdeb), and mona monkeys (Cercopithecus mona, SIVmon). Briefly, SIV isolates segregate phylogenetically based upon their species of origin into at least five lineages represented by SIVsm, SIVagm, SIVsyk, SIVlhoest, and SIVcpz. SIVsm forms a genetic group with human immunodeficiency virus type 2 (HIV-2) and SIVmac from captive macaques, and SIVcpz clusters with HIV-1 (44, 45), consistent with the origins of HIV in African primates (16). Some of the SIV strains, such as SIVrcm, SIVgsn, SIVdrl, SIVmnd type 2, and SIVcpz are obviously recombinant. There is also evidence of cross-species transmission between African primates such as drills and mandrills (28) and patas monkeys and AGM (7), suggesting a long and complex evolutionary history of the primate lentiviruses. Of all of the SIVs, those isolated from wild-caught AGM have been the most thoroughly characterized (3, 15, 25, 29, 30, 38). SIVagm isolates form a lineage that consists of four distinct subtypes that cluster depending upon the species of AGM from which they were isolated: grivets (Chlorocebus aethiops, SIVagm/gri) (15), vervets (Chlorocebus pygerythrus, SIVagm/ver) (1, 30), sabaeus (Chlorocebus sabaeus, SIVagm/sab) (29), and tantalus (Chlorocebus tantalus, SIVagm/tan) (25, 38, 54).
Although natural infection is widespread in feral populations of AGMs (31) and sooty mangabeys (17), this infection does not result in immunodeficiency (10, 24, 37). However, experimental inoculation of Asian macaque species results in an immunodeficiency syndrome that is remarkably similar in pathogenesis to human AIDS (6, 12, 34, 53). Thus, SIVsm infection of macaques results in AIDS, and similarly, experimental inoculation of pigtailed (PT) macaques with at least one SIVagm isolate results in AIDS (22). SIVagm isolated from a naturally infected African green monkey (AGM 90) of the vervet species (C. pygerythrus) was found to induce AIDS in one (PT63) of two inoculated PT macaques (19, 22). Virus isolated from the SIVagm-infected PT macaque that developed AIDS, designated SIVagm9063 to indicate the two monkeys that gave rise to this variant (AGM 90 and PT63), was uniformly pathogenic upon experimental inoculation of PT macaques (22). It is not clear, however, whether this macaque-passaged isolate was pathogenic due to evolution and adaptation in the macaque or if the original natural vervet isolate was equivalently pathogenic. In addition, it is unclear whether this isolate is unique in terms of its virulence in macaques or whether other natural SIVagm isolates may also induce AIDS in PT macaques. Another natural vervet isolate was reported to induce AIDS in macaques but only following coinfection with simian type D retrovirus (20). Many of the studies of pathogenicity of SIVagm with macaques have failed to show evidence of disease (24, 37). However, the majority of studies have used rhesus or cynomolgus macaques rather than pigtailed macaques (37). Since studies with SIVagm9063 demonstrated that this virus did not replicate efficiently in rhesus macaques and was not pathogenic in this species (22), PT macaques may be required to adequately show the pathogenic potential of these viruses.
The comparative study of the pathogenesis of such viruses in their natural host and a disease-susceptible host such as macaques may shed light on the pathogenesis of AIDS in humans. The SIV strains that have been the best characterized in terms of pathogenesis in macaques are SIVsm and the related SIVmac strains (5, 6, 24), (53). The semi-steady-state level at which plasma viremia plateaus after the primary phase of infection is an excellent predictor of subsequent disease course in SIVsm- and SIVmac-infected macaques (18, 33, 56, 61). Macaques with persistent high plasma viremia succumb more rapidly to disease than those with lower levels of plasma viremia. This correlation suggests that the extent of viral replication is a major determinant of disease pathogenesis. A similar association has been observed in HIV-1-infected humans (35, 36). Based upon this paradigm, one would have expected naturally infected animals to exhibit low levels of viremia. In reality, AGMs, sooty mangabeys, mandrills, and chimpanzees infected naturally with their own unique viruses display moderate to high levels of plasma viremia (8, 10, 13, 19, 27, 42, 46, 52). Preliminary studies with SIVagm infection of PT macaques demonstrated high levels of plasma viremia, as assessed by p27 antigen capture assays and, more recently, with real time reverse transcription (RT)-PCR assays (22). However, the steady-state levels of viremia in PT macaques have not been studied to determine whether this pathogenic infection also exhibits the same correlations of viral load with disease progression. The purpose of the present study was to determine the pathogenicity of two natural isolates of SIVagm (SIVagm90 and SIVagm155) in PT macaques. A secondary and equally critical goal was to assess whether plateau levels of viremia correlated with disease progression in SIVagm, as in other previously studied pathogenic primate lentivirus infections.
MATERIALS AND METHODS
Animals and viruses. The naturally infected African green monkeys that were the source of SIVagm90 and SIVagm155 were imported from Kenya in 1987 and phenotypically appear to be of the vervet species (C. pygerythrus). Virus load and tissue distribution were studied previously in these animals (19, 22). Virus was isolated from the mesenteric lymph nodes of AGM90 and AGM155 by coculture of viably frozen mononuclear cells with macaque peripheral blood mononuclear cells (PBMC) (19). The macaque-passaged derivative, SIVagm9063, was isolated from the spleen of PT63 during necropsy at the time of terminal AIDS, as previously described (22). Viruses were titrated for infectivity in the susceptible cell line CEMss, and groups of four PT macaques were inoculated intravenously with 1,000 50% tissue culture infective doses of the respective viruses. Animals were maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals (40) and were housed in a biosafety level 2 facility; biosafety level 3 practices were followed (40).
Cloning and sequence analysis of envelopes. The complete sequence of SIVagm9063 and SIVagm155 have been previously reported (22, 30). SIVagm155-4 was cloned from a long-term passage of virus in rhesus macaque PBMC that was isolated from PBMC of AGM155. This was a different isolate of SIVagm155 than that used for the present studies. Full-length envelope clones of the SIVagm90 isolate were derived by PCR amplification with primers 3688-F (5'-AAGAGGACCTTAGGTGATTACCTATCATGCC-3') and 3689-R (5'-CCCTTTTCTTTTAAAAAGTGCGAAAA-3') to amplify a 2,878-bp fragment. Bands of the correct size were excised, cloned into pCRII-TOPO vector (TOPO TA cloning kit; Invitrogen, Carlsbad, Calif.), and sequenced by automated fluorescent sequencing (Taq amplification/termination; Perkin-Elmer Applied Biosystems, Warrington, United Kingdom). Sequences were aligned by using the Geneworks (Intelligenetics, Campbell, Calif.) sequence analysis program to compile alignments of both nucleotide and amino acid sequences.
Real-time plasma viral RNA assay. A real time RT-PCR assay for quantitation of viral RNA in plasma was performed as previously described (19) by using a method based on the Prism 770 sequence detection system (Applied Biosystems, Foster City, Calif.) that was used previously for SIVsm/mac-specific real-time PCR (19, 57). An alignment of SIVagm full-length sequences (SIVagm155-4, SIVagmTYO-1, SIVagm3, and SIVagm9063-2) from vervets was used to identify a highly conserved region within the transmembrane glycoprotein-encoding region of envelope. The forward and reverse primers to amplify a 122-nucleotide fragment and an internal fluorogenic probe primer were generated by using inosine (i) residues at positions that were not conserved between SIVagm sequences. The sequences were as follows: AgmF, 5'-GTC CAG TCT CiG CAi TTi CTT G-3'; AgmR, 5'-CGG GCA TTG AGG TTT TTi AC-3'; probe, 5'-FAM-CAG iTG TTG AAG CTi ACC ATT TGG G-TAMRA-3. A high-performance liquid chromatography-purified probe was obtained from Applied Biosystems. A purified in vitro sense RNA runoff transcript corresponding to 1.9-kb fragment (HindIII/HincII) of the SIVagm9063 envelope was used to develop a standard curve for the assay. Plasma samples for analysis were collected by using EDTA as an anticoagulant and stored in a –80°C freezer until analysis.
Lymphocyte immunophenotyping. Lymphocyte subsets (CD4, CD2, CD8, and B cells) were analyzed sequentially throughout the infection by fluorescence-activated cell sorter analysis by FAST Systems, Inc., Gaithersburg, Md. Briefly, heparinized whole-blood samples were incubated for 20 min in the dark at 4°C in the presence of sodium azide with the appropriate monoclonal antibody conjugated to fluorochrome. For CD4 lymphocytes, CD4 (phycoerythrin) (Exalpha Biological, Inc., Boston, Mass.) and CD2 (fluorescein isothiocyanate) (Genetrak, Inc., Liberty, N.C.), and for CD8 lymphocytes, CD8 (phycoerythrin) and CD20 (fluorescein isothiocyanate) (Becton Dickinson, San Jose, Calif.) were used to identify CD20, expressed on B lymphocytes. Following staining, erythrocytes were lysed and leukocytes were fixed in 1% paraformaldehyde and analyzed with a Coulter Epics 753.
SIV-specific antibodies. Serology for antibodies to SIVagm was performed by Western blot analysis, as previously described (21). Briefly, virus was pelleted from the cell-free supernatant of CEMss cells infected with SIVagm90. Virus particles were disrupted in Laemmli sample buffer, and viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Individual strips containing SIVagm viral proteins were reacted with diluted macaque plasma and washed to remove unbound material. The bound SIV-specific antibodies were visualized by subsequent reaction with ImmunoPure A/G protein conjugated with alkaline phosphatase (Pierce Biotechnology, Rockford, Ill.), followed by the nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate system (KPL, Gaithersburg, Md.).
SIV-specific ISH. Nonradioactive in situ hybridization (ISH) for SIV expression was performed in formalin-fixed, paraffin-embedded lymph nodes utilizing sense or antisense digoxigenin-labeled riboprobes that spanned the entire SIVagm9063-2 genome as previously described (22). Briefly, sections were deparaffinized and rehydrated with water, pretreated with 0.2 N HCl and proteinase K, prehybridized, and then hybridized overnight al 51°C with either the antisense or the sense riboprobe. The riboprobe consisted of a mixture of probes encompassing 90% of the SIVagm9063 genome conjugated with digoxigenin-UTP (Lofstrand Labs Ltd., Gaithersburg, Md.) at a final concentration of 1.75 ng/μl. The hybridized sections were washed in standard posthybridization buffers and RNase solutions (RnaseA [Sigma] and RNaseT1 [Roche Molecular Biochemicals, Indianapolis. Ind.]). The sections were blocked in 3% normal sheep and horse serum in 0.1 M Tris (pH 7.4) and then incubated with a 1:500 dilution of sheep anti-digoxigenin-alkaline phosphatase (Roche) for 1 h. Sections were then rinsed in Tris buffer and reacted with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Vector Laboratories, Ltd., Burlingame, Calif.) for 10 h and visualized with an Axiophot microscope (Carl Zeiss, Inc., Thornwood. N.Y.).
RESULTS
Description of virus stocks. A total of 11 pigtailed macaques were inoculated intravenously with equivalent tissue culture infective doses of three SIVagmVer isolates, SIVagm90, SIVagm9063, and SIVagm155. These cell-free virus stocks were generated by growth in PBMC from a common PT macaque donor. Four macaques were inoculated with SIVagm90, a virus that had been isolated from the mesenteric lymph node of the naturally infected, wild-caught African green monkey imported from Kenya in 1987 (AGM90). Four macaques were inoculated with our previously described pathogenic SIVagm9063, an isolate from a pigtailed macaque inoculated with SIVagm90 (22). An additional three macaques were inoculated with SIVagm155 that had been isolated from the lymph node of AGM155 (19), an AGM from the same cohort as AGM90.
In terms of genetic interrelationships of these viruses, infectious molecular clones of SIVagm9063 and SIVagm155 have been previously described (22, 30). SIVagm155-4 was cloned from a PBMC isolate from AGM155 that had been passaged extensively in rhesus PBMC and thus is not entirely representative of the isolate from lymphoid tissues used in the present study. These viruses cluster within the vervet lineage of SIVagm, sharing 88, 83, and 80% identity, respectively, in Gag, Pol, and Env sequences. SIVagm9063 was isolated after in vivo passage of SIVagm90 in pigtailed macaque PT63 and, therefore, was presumed to have diverged genetically from the parental virus. To evaluate the degree of divergence of these two viruses, envelope sequences of SIVagm90 were cloned following RT-PCR amplification from the virus stock used for macaque inoculation. Envelopes derived from the SIVagm90 stock were closely related to one another (95% identity) and slightly more divergent from SIVagm9063 (90% identity) but significantly closer genetically than to SIVagm155. As shown in Fig. 1, variable residues were clustered in defined variable regions. The majority of potential N-linked glycosylation sites were conserved (n = 18), and all cysteine residues were conserved among the three strains. Distinct variable regions were observed with insertion/deletion polymorphisms in the V1 region. The cysteine loop that is analogous to the HIV-1 V3 loop (residues 274 to 307) was generally well conserved, consistent with previous observations on this region of SIVagm and SIVsm. However, a region of hypervariability was observed immediately C-terminal to this loop. Variable regions (V4 and V5) flanked the previous defined and highly conserved CD4-binding domain. The envelope clones of SIVagm90 and SIVagm9063 encoded full-length transmembrane (TM) proteins, whereas the TM protein of the molecularly cloned SIVagm155-4 was prematurely truncated in the cytoplasmic tail region. SIVagm155-4 was derived from a culture that had been passaged extensively in rhesus macaque PBMC, a procedure often associated with tissue culture adaptation. Macaques in the present study were inoculated with a virus isolated from tissues of AGM155 by short-term culture with a PT macaque PBMC isolate that encoded a full-length TM glycoprotein (data not shown).
Variable plasma viremia among SIVagm-infected macaques. Following intravenous inoculation, the macaques were monitored sequentially by virus isolation from PBMC, flow cytometric analysis of CD4+ and CD8+ T cells and B cells in blood, Western blot analysis of plasma for antibody responses, SIV-specific in situ hybridization of lymph node biopsy specimens, and plasma viral RNA loads. All of the animals became infected and seroconverted, as shown by Western blot analysis (data not shown). As shown in Table 1, infectious virus was successfully isolated from PBMC of each of the inoculated macaques during the early phases of infection. Virus was also recovered from lymph node mononuclear cells of each macaque at 1, 2, 4, and 8 weeks postinoculation (data not shown). Virus isolation from PBMC became less consistent at later time points in some of the animals (PT9636, PT9665, PT9657, PT9669, and PT7119), suggestive of low viral loads in these animals. This was particularly obvious in macaques inoculated with SIVagm155.
Plasma viremia was monitored sequentially throughout the course of infection by using real-time RT-PCR with SIVagmVer-specific primers and probe (19). As demonstrated in Fig. 2, high levels of primary plasma viremia were achieved in all animals by 1 week after inoculation (ranging from 107 to 108/ml of plasma), regardless of virus inoculum. Viral RNA loads in plasma declined sharply in all of the animals to varying degrees, reaching a semi-steady-state plateau (or setpoint) by approximately 8 to 12 weeks postinfection. The plateau levels varied widely between animals from 102 to 106 per ml, with the widest range among animals inoculated with SIVagm90. The plasma viral load setpoint was consistently high in SIVagm9063-inoculated macaques and consistently lower in SIVagm155-inoculated macaques. Four animals demonstrated good control of viremia to levels generally less than 1,000 copies/ml; these macaques included one inoculated with SIVagm90 and all three inoculated with SIVagm155. This was in complete agreement with the general difficulty in isolation of virus from PBMC (Table 1) of these animals. In contrast, virus isolation was consistently successful from animals with high plateau levels of plasma viremia.
Lymph node biopsy specimens sampled at 1, 2, and 4 weeks postinoculation were evaluated by SIVagm-specific in situ hybridization. The numbers of SIV-expressing cells observed in lymph node biopsy specimens was expressed as the average of the number of positive cells in 10 high power fields (40x objective), and the kinetics of virus expression in each of the animals is shown graphically in the top of Fig. 3. Large numbers of SIV-expressing cells were observed in 1-week biopsy specimens, concurrent with the peak of plasma viremia, and the numbers of SIV-positive cells declined by 2 weeks, with evidence of diffuse staining of germinal centers of follicles by 4 weeks postinoculation (see bottom of Fig. 3). As shown, peak virus expression was generally observed at 1 week postinfection (p.i.), coincident with peak plasma viremia, and subsequently declined to low but still detectable numbers of infected cells by 4 weeks p.i. Virus expression was extremely rare in lymph nodes biopsied from animals inoculated with SIVagm155. Thus, virus-expressing cells were only observed in lymph nodes sampled from this group of animals at 1 week and one from 2 weeks. The number of SIV-expressing cells was also lower than in SIVagm90- and SIVagm9063-infected macaques, and trapping of virus in germinal centers was not evident.
SIVagm90 infection results in blood CD4+-T-cell depletion. Lymphocyte subsets in the blood were monitored sequentially throughout infection. As shown in Fig. 4 and 5, a decline in CD4+ and CD8+ T cells and B cells was observed in all animals coincident with the peak of plasma viremia (i.e., 7 to 10 days p.i.). These lymphocyte subsets generally rebounded to near preinoculation values in the majority of animals. However, progressive CD4 depletion leading to CD4+-T-cell numbers of <100 CD4+ T cells/μl by approximately 6 months p.i. was observed in two of SIVagm90- and three SIVagm9063-inoculated PTs, respectively. Less-pronounced CD4 depletion was observed in the other animals in these two cohorts. In contrast, CD4+ T cells in the blood of SIVagm155-inoculated PTs remained relatively stable (Fig. 5). However, even in these three animals, the levels CD4+ T cells remained significantly lower than preinoculation values, with a slower trend toward loss of CD4+ T cells over time from inoculation (Fig. 4). CD8+-T-cell and B cell numbers also declined but never to the same degree as observed in the CD4+-T-cell subset. Some animals showed transient increases in B cell numbers (PT9657 and PT9669) or CD8 numbers (PT9668). Immunohistochemical analysis of lymph nodes, spleens, and gastrointestinal lymphoid tissues collected terminally in macaques with peripheral CD4-T-cell depletion confirmed a generalized depletion of CD4 T cells in lymphoid tissues (data not shown).
SIVagm90 and SIVagm9063 induce AIDS in PT macaques. Macaques were euthanized if they lost >20% body weight or had persistent diarrhea or other clinical symptoms that were not responsive to supportive and antibiotic therapy. As shown in Table 2, three animals inoculated with SIVagm90 and three animals inoculated with SIVagm9063 were euthanized over the first 2 years following inoculation. These animals included all the animals with severe CD4 depletion as well as one other (PT9636) that died due to thrombosis of the pulmonary artery, a complication we have observed previously in SIV-infected PT macaques (24). As detailed in Table 2, many of these animals had pathological evidence of severe enteritis with blunting of villus tips, lymphoid depletion in gastrointestinal-associated lymphoid tissues, edema, and crypt abscessation, although the etiologic agents were not identified. Three of the animals had bacterial infections, fulminant necrotizing bacterial pancreatitis and peritonitis (PT9529), bacterial pneumonia (PT9523), and disseminated lymphadenitis and enteritis due to Mycobacterium avium complex infection. Other AIDS-related pathological findings included membranoproliferative glomerulonephritis and pulmonary thrombosis. In situ hybridization for SIV expression in tissues collected terminally from these animals demonstrated a range in virus loads among animals, with the highest levels observed in PT9414 and PT9515 (data not shown). In contrast, all of the macaques inoculated with SIVagm155 remain clinically healthy at 140 weeks postinoculation.
Inverse correlation between plasma viral setpoint and CD4+-T-cell depletion. Since viral load and outcome varied greatly among these PT macaques, the correlation between the plasma viral setpoint and disease outcome was evaluated by using the Pearson correlation coefficient. We evaluated the correlation between the viral setpoint and various measures of disease outcomes including survival, slope of CD4+-T-cell loss, and the percentage of CD4 cells by 6 months postinoculation compared to preinoculation. Plasma viral load at setpoint was defined operationally as the viral RNA load at 12 weeks postinoculation. The percent CD4 loss by 6 months of infection correlated with survival (r = 0.782, P < 0.0001) and with the slope of CD4 loss over the 140 weeks of observation (r = 0.768, P = 0.0007), and therefore, since this has greater value in evaluating disease progression during infection, we used the percentage of CD4 cells remaining at 6 months as a general measure of disease progression. As shown in Fig. 6, a statistically significant inverse correlation was observed between the log10 plasma viral load at the setpoint with the percent loss of CD4+ T cells at 6 weeks postinfection (r = –0.951, P < 0.0001). Thus, the higher the plasma viral load, the greater the CD4+-T-cell depletion observed. The viral setpoint also correlated inversely with survival (r = –0.742, P < 0.01).
Cursory inspection of virus load and CD4 loss in these animals suggested that SIVagm155 was less pathogenic than either SIVagm90 or SIVagm9063. We therefore used Student's t test to compare peak viral load (log10), setpoint viral load, and the percentage of CD4 T cells at 6 months postinoculation among the three groups of animals. As shown in Table 3, the peak viral load did not differ significantly among the three groups. In addition, setpoint viremia and CD4 loss did not differ significantly between SIVagm90- and SIVagm9063-inoculated macaques (P = 0.739 and P = 0.402, respectively). However, setpoint viremia was significantly higher in SIVagm90/9063-inoculated macaques than in SIVagm155-inoculated macaques (P = 0.0006), and these latter animals also maintained significantly higher levels of CD4+ T cells than SIVagm90/9063-infected PTs (P = 0.004). However, despite reduced virulence of SIVagm155 and apparent clinical health, these animals do not appear to be immunologically normal. They all exhibit some degree of lymphadenopathy, and their CD4+-T-cell numbers are reduced compared to preinoculation values, suggesting that, even at low virus loads, SIVagm infection has pathological consequences in PT macaques.
DISCUSSION
The present study confirms the pathogenicity of SIVagm90 and a macaque-passaged derivative (SIVagm9063) in PT macaques. Infection of PT macaques with either of these virus isolates resulted in the progressive loss of circulating CD4+ T lymphocytes, severe lymphoid depletion, and the development of an AIDS-like syndrome. Macaque passage of this virus was not required for pathogenicity, although the virus was not uniformly pathogenic in this small cohort of macaques. We therefore conclude that this natural virus isolate is fully competent to induce AIDS in macaques without genetic and biologic selection in this foreign host species. Since SIVagm90 was isolated in macaque PBMC, we cannot rule out the possibility that growth in macaque PBMC results in some selection of already-existing members of the viral quasispecies. This study also clearly demonstrates that not all natural isolates of SIVagm are equally pathogenic; infection of PT macaques with the related SIVagm155 was rapidly controlled and did not induce AIDS in a period of observation of 140 weeks. Pathogenic and nonpathogenic SIVagm infection of macaques could not be distinguished by the levels of primary viremia, which was robust in all inoculated animals, but disease progression was associated with persistent plasma viremia.
Pathogenic infection of macaques with SIVagm appeared to be similar to that of SIVsm/mac infection of macaques, with perhaps a slower rate of disease progression. These animals developed lymphadenopathy, progressive CD4+-T-cell decline with the development of terminal opportunistic infections and showed a similar disease course and kinetics of primary and setpoint viremia. Critically, SIVagm was similar to SIVmac/sm infection in the demonstration of an inverse correlation of virus load in plasma with disease course. Thus, the mechanisms of AIDS induction are likely to be common between these pathogenic virus infections. Although there are clearly aspects of SIVagm infection that have not yet been explored, such as the generation of cytotoxic T cell and neutralization escape mutants or effects on CCR5-expressing memory CD4 T cells in mucosal sites, we conclude that SIVagm infection of PT macaques is a valid animal model to explore pathogenic mechanisms of human AIDS.
While SIVagm-PT macaque model is not a novel model for AIDS, the ability to compare and contrast pathogenic (macaque) and nonpathogenic (AGM) infection in different host species with a common virus strain may give unique insights into the pathogenesis of AIDS in humans. Despite years of study, it is apparent that the mechanisms underlying immunodeficiency in HIV and SIV infection are poorly understood. The correlation between plasma viremia and rapidity of disease progression in pathogenic SIV and HIV infections suggests that much of the CD4 depletion that occurs in AIDS is the direct result of virus-induced cell death. Initially, AIDS was assumed to be due to direct killing of CD4+ T cells, an obvious conclusion based upon the fact that HIV and SIV use CD4 as their cellular receptor. However, AIDS can occur in both humans and macaques in the face of incomplete CD4+-T-cell depletion (26), suggesting qualitative dysfunction of CD4+ T cells or specific subset depletion. Indeed, earlier studies of SIVmac infection showed early selective depletion of CCR5+, memory CD4+ T cells in the gastrointestinal tract and other mucosal systems (60). A recent study of SIVsm-infected macaques revealed a similar early depletion of CCR5+ memory cells in the peripheral blood; this contrasted with depletion of CXCR4+ naive cells in simian human immunodeficiency virus infection (41). These data collectively suggest that much of the early immune dysfunction in SIV and HIV infection occurs by depletion of a subset of CD4+-T-cell populations such that the host is never able to gain advantage over the virus infection. SIV-infected macaques survive for as long as 2 years after infection with very gradual deterioration of the immune system, suggesting a complex interplay between the virus and the immune system that involves early destruction of memory T cells by virus, followed by increased overall memory T cell turnover, thymic dysfunction, and limitations in peripheral T cell regeneration (14).
The comparative study of pathogenic and nonpathogenic (natural) models of SIV infection is likely to give additional insight into the mechanisms of AIDS. Since the correlation between viral replication levels and disease have been a constant theme in SIV infection of macaques and HIV infection of humans, the initial assumption regarding the lack of disease in natural host species was that it resulted from strict containment of viral replication, analogous to what is observed with long-term nonprogressors of HIV infection (9). However, this is clearly not the case. Naturally infected AGM (8, 19, 27), sooty mangabeys (10, 11, 32, 52), and mandrills (42, 58) appear to have significant, ongoing viral replication, similar to levels during the asymptomatic phase of pathogenic infection in macaques and humans. Genetic evolution of SIV in both AGM and sooty mangabeys also suggests ongoing, persistent viral replication (10, 39, 46). Thus, natural models of SIV are not analogous in virologic terms to long-term nonprogressors of HIV infection. Studies with this virus in AGM demonstrate robust virus replication (unpublished data) comparable to levels of viremia observed in naturally infected AGM. This present study demonstrates that the natural SIVagm90 isolate would be appropriate for comparative studies of SIVagm in PT and AGM. We believe it is critical to use a common virus in these two species for comparative purposes to allow the use of common assays and reagents. The use of SIVagm90 in AGM would be appropriate for modeling natural infection of AGM, since it is not adapted to macaques, and will be the focus of future studies.
ACKNOWLEDGMENTS
We thank C. Erb, S. Whitted, and R. Goeken of LMM, NIAID, for technical assistance and M. A. Robinson and T. Myers of RTB, NIAID, for assistance on statistical analysis.
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Bioqual, Inc., Rockville, Maryland
ABSTRACT
Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques (Macaca nemestrina) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4+-T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+-T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4+-T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (P values of <0.01). Considering all the macaques, the degree of CD4+-T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.
INTRODUCTION
The simian immunodeficiency viruses (SIV) are a diverse group of viruses that naturally infect a wide range of African primates (2, 21, 43, 49). At the present time, SIV from at least seven African monkey species have been identified and molecularly characterized. The genetic relationships among many of these SIV strains have been reviewed previously (2, 43, 49-51). Full-length sequences have currently been characterized from sooty mangabey monkeys (Cercocebus atys, SIVsm), mandrills and drills (Mandrillus sphinx and Mandrillus leucophaeus, SIVmnd and SIVdrl) (55, 58, 59), Sykes monkeys (Cercopithecus albogularis mitis, SIVsyk) (23), African green monkeys (AGM), (Chlorocebus sp., SIVagm), chimpanzees (Pan troglodytes, SIVcpz) (4, 16, 47, 48), redcapped mangabeys (Cercocebus torquatus, SIVrcm), colobus monkeys (Colobus guerza, SIVcol), and a number of African guenons, including greater spot-nosed guenons (Cercopithecus nictitans, SIVgsn), DeBrazza monkeys (Cercopithecus neglectus, SIVdeb), and mona monkeys (Cercopithecus mona, SIVmon). Briefly, SIV isolates segregate phylogenetically based upon their species of origin into at least five lineages represented by SIVsm, SIVagm, SIVsyk, SIVlhoest, and SIVcpz. SIVsm forms a genetic group with human immunodeficiency virus type 2 (HIV-2) and SIVmac from captive macaques, and SIVcpz clusters with HIV-1 (44, 45), consistent with the origins of HIV in African primates (16). Some of the SIV strains, such as SIVrcm, SIVgsn, SIVdrl, SIVmnd type 2, and SIVcpz are obviously recombinant. There is also evidence of cross-species transmission between African primates such as drills and mandrills (28) and patas monkeys and AGM (7), suggesting a long and complex evolutionary history of the primate lentiviruses. Of all of the SIVs, those isolated from wild-caught AGM have been the most thoroughly characterized (3, 15, 25, 29, 30, 38). SIVagm isolates form a lineage that consists of four distinct subtypes that cluster depending upon the species of AGM from which they were isolated: grivets (Chlorocebus aethiops, SIVagm/gri) (15), vervets (Chlorocebus pygerythrus, SIVagm/ver) (1, 30), sabaeus (Chlorocebus sabaeus, SIVagm/sab) (29), and tantalus (Chlorocebus tantalus, SIVagm/tan) (25, 38, 54).
Although natural infection is widespread in feral populations of AGMs (31) and sooty mangabeys (17), this infection does not result in immunodeficiency (10, 24, 37). However, experimental inoculation of Asian macaque species results in an immunodeficiency syndrome that is remarkably similar in pathogenesis to human AIDS (6, 12, 34, 53). Thus, SIVsm infection of macaques results in AIDS, and similarly, experimental inoculation of pigtailed (PT) macaques with at least one SIVagm isolate results in AIDS (22). SIVagm isolated from a naturally infected African green monkey (AGM 90) of the vervet species (C. pygerythrus) was found to induce AIDS in one (PT63) of two inoculated PT macaques (19, 22). Virus isolated from the SIVagm-infected PT macaque that developed AIDS, designated SIVagm9063 to indicate the two monkeys that gave rise to this variant (AGM 90 and PT63), was uniformly pathogenic upon experimental inoculation of PT macaques (22). It is not clear, however, whether this macaque-passaged isolate was pathogenic due to evolution and adaptation in the macaque or if the original natural vervet isolate was equivalently pathogenic. In addition, it is unclear whether this isolate is unique in terms of its virulence in macaques or whether other natural SIVagm isolates may also induce AIDS in PT macaques. Another natural vervet isolate was reported to induce AIDS in macaques but only following coinfection with simian type D retrovirus (20). Many of the studies of pathogenicity of SIVagm with macaques have failed to show evidence of disease (24, 37). However, the majority of studies have used rhesus or cynomolgus macaques rather than pigtailed macaques (37). Since studies with SIVagm9063 demonstrated that this virus did not replicate efficiently in rhesus macaques and was not pathogenic in this species (22), PT macaques may be required to adequately show the pathogenic potential of these viruses.
The comparative study of the pathogenesis of such viruses in their natural host and a disease-susceptible host such as macaques may shed light on the pathogenesis of AIDS in humans. The SIV strains that have been the best characterized in terms of pathogenesis in macaques are SIVsm and the related SIVmac strains (5, 6, 24), (53). The semi-steady-state level at which plasma viremia plateaus after the primary phase of infection is an excellent predictor of subsequent disease course in SIVsm- and SIVmac-infected macaques (18, 33, 56, 61). Macaques with persistent high plasma viremia succumb more rapidly to disease than those with lower levels of plasma viremia. This correlation suggests that the extent of viral replication is a major determinant of disease pathogenesis. A similar association has been observed in HIV-1-infected humans (35, 36). Based upon this paradigm, one would have expected naturally infected animals to exhibit low levels of viremia. In reality, AGMs, sooty mangabeys, mandrills, and chimpanzees infected naturally with their own unique viruses display moderate to high levels of plasma viremia (8, 10, 13, 19, 27, 42, 46, 52). Preliminary studies with SIVagm infection of PT macaques demonstrated high levels of plasma viremia, as assessed by p27 antigen capture assays and, more recently, with real time reverse transcription (RT)-PCR assays (22). However, the steady-state levels of viremia in PT macaques have not been studied to determine whether this pathogenic infection also exhibits the same correlations of viral load with disease progression. The purpose of the present study was to determine the pathogenicity of two natural isolates of SIVagm (SIVagm90 and SIVagm155) in PT macaques. A secondary and equally critical goal was to assess whether plateau levels of viremia correlated with disease progression in SIVagm, as in other previously studied pathogenic primate lentivirus infections.
MATERIALS AND METHODS
Animals and viruses. The naturally infected African green monkeys that were the source of SIVagm90 and SIVagm155 were imported from Kenya in 1987 and phenotypically appear to be of the vervet species (C. pygerythrus). Virus load and tissue distribution were studied previously in these animals (19, 22). Virus was isolated from the mesenteric lymph nodes of AGM90 and AGM155 by coculture of viably frozen mononuclear cells with macaque peripheral blood mononuclear cells (PBMC) (19). The macaque-passaged derivative, SIVagm9063, was isolated from the spleen of PT63 during necropsy at the time of terminal AIDS, as previously described (22). Viruses were titrated for infectivity in the susceptible cell line CEMss, and groups of four PT macaques were inoculated intravenously with 1,000 50% tissue culture infective doses of the respective viruses. Animals were maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals (40) and were housed in a biosafety level 2 facility; biosafety level 3 practices were followed (40).
Cloning and sequence analysis of envelopes. The complete sequence of SIVagm9063 and SIVagm155 have been previously reported (22, 30). SIVagm155-4 was cloned from a long-term passage of virus in rhesus macaque PBMC that was isolated from PBMC of AGM155. This was a different isolate of SIVagm155 than that used for the present studies. Full-length envelope clones of the SIVagm90 isolate were derived by PCR amplification with primers 3688-F (5'-AAGAGGACCTTAGGTGATTACCTATCATGCC-3') and 3689-R (5'-CCCTTTTCTTTTAAAAAGTGCGAAAA-3') to amplify a 2,878-bp fragment. Bands of the correct size were excised, cloned into pCRII-TOPO vector (TOPO TA cloning kit; Invitrogen, Carlsbad, Calif.), and sequenced by automated fluorescent sequencing (Taq amplification/termination; Perkin-Elmer Applied Biosystems, Warrington, United Kingdom). Sequences were aligned by using the Geneworks (Intelligenetics, Campbell, Calif.) sequence analysis program to compile alignments of both nucleotide and amino acid sequences.
Real-time plasma viral RNA assay. A real time RT-PCR assay for quantitation of viral RNA in plasma was performed as previously described (19) by using a method based on the Prism 770 sequence detection system (Applied Biosystems, Foster City, Calif.) that was used previously for SIVsm/mac-specific real-time PCR (19, 57). An alignment of SIVagm full-length sequences (SIVagm155-4, SIVagmTYO-1, SIVagm3, and SIVagm9063-2) from vervets was used to identify a highly conserved region within the transmembrane glycoprotein-encoding region of envelope. The forward and reverse primers to amplify a 122-nucleotide fragment and an internal fluorogenic probe primer were generated by using inosine (i) residues at positions that were not conserved between SIVagm sequences. The sequences were as follows: AgmF, 5'-GTC CAG TCT CiG CAi TTi CTT G-3'; AgmR, 5'-CGG GCA TTG AGG TTT TTi AC-3'; probe, 5'-FAM-CAG iTG TTG AAG CTi ACC ATT TGG G-TAMRA-3. A high-performance liquid chromatography-purified probe was obtained from Applied Biosystems. A purified in vitro sense RNA runoff transcript corresponding to 1.9-kb fragment (HindIII/HincII) of the SIVagm9063 envelope was used to develop a standard curve for the assay. Plasma samples for analysis were collected by using EDTA as an anticoagulant and stored in a –80°C freezer until analysis.
Lymphocyte immunophenotyping. Lymphocyte subsets (CD4, CD2, CD8, and B cells) were analyzed sequentially throughout the infection by fluorescence-activated cell sorter analysis by FAST Systems, Inc., Gaithersburg, Md. Briefly, heparinized whole-blood samples were incubated for 20 min in the dark at 4°C in the presence of sodium azide with the appropriate monoclonal antibody conjugated to fluorochrome. For CD4 lymphocytes, CD4 (phycoerythrin) (Exalpha Biological, Inc., Boston, Mass.) and CD2 (fluorescein isothiocyanate) (Genetrak, Inc., Liberty, N.C.), and for CD8 lymphocytes, CD8 (phycoerythrin) and CD20 (fluorescein isothiocyanate) (Becton Dickinson, San Jose, Calif.) were used to identify CD20, expressed on B lymphocytes. Following staining, erythrocytes were lysed and leukocytes were fixed in 1% paraformaldehyde and analyzed with a Coulter Epics 753.
SIV-specific antibodies. Serology for antibodies to SIVagm was performed by Western blot analysis, as previously described (21). Briefly, virus was pelleted from the cell-free supernatant of CEMss cells infected with SIVagm90. Virus particles were disrupted in Laemmli sample buffer, and viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Individual strips containing SIVagm viral proteins were reacted with diluted macaque plasma and washed to remove unbound material. The bound SIV-specific antibodies were visualized by subsequent reaction with ImmunoPure A/G protein conjugated with alkaline phosphatase (Pierce Biotechnology, Rockford, Ill.), followed by the nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate system (KPL, Gaithersburg, Md.).
SIV-specific ISH. Nonradioactive in situ hybridization (ISH) for SIV expression was performed in formalin-fixed, paraffin-embedded lymph nodes utilizing sense or antisense digoxigenin-labeled riboprobes that spanned the entire SIVagm9063-2 genome as previously described (22). Briefly, sections were deparaffinized and rehydrated with water, pretreated with 0.2 N HCl and proteinase K, prehybridized, and then hybridized overnight al 51°C with either the antisense or the sense riboprobe. The riboprobe consisted of a mixture of probes encompassing 90% of the SIVagm9063 genome conjugated with digoxigenin-UTP (Lofstrand Labs Ltd., Gaithersburg, Md.) at a final concentration of 1.75 ng/μl. The hybridized sections were washed in standard posthybridization buffers and RNase solutions (RnaseA [Sigma] and RNaseT1 [Roche Molecular Biochemicals, Indianapolis. Ind.]). The sections were blocked in 3% normal sheep and horse serum in 0.1 M Tris (pH 7.4) and then incubated with a 1:500 dilution of sheep anti-digoxigenin-alkaline phosphatase (Roche) for 1 h. Sections were then rinsed in Tris buffer and reacted with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Vector Laboratories, Ltd., Burlingame, Calif.) for 10 h and visualized with an Axiophot microscope (Carl Zeiss, Inc., Thornwood. N.Y.).
RESULTS
Description of virus stocks. A total of 11 pigtailed macaques were inoculated intravenously with equivalent tissue culture infective doses of three SIVagmVer isolates, SIVagm90, SIVagm9063, and SIVagm155. These cell-free virus stocks were generated by growth in PBMC from a common PT macaque donor. Four macaques were inoculated with SIVagm90, a virus that had been isolated from the mesenteric lymph node of the naturally infected, wild-caught African green monkey imported from Kenya in 1987 (AGM90). Four macaques were inoculated with our previously described pathogenic SIVagm9063, an isolate from a pigtailed macaque inoculated with SIVagm90 (22). An additional three macaques were inoculated with SIVagm155 that had been isolated from the lymph node of AGM155 (19), an AGM from the same cohort as AGM90.
In terms of genetic interrelationships of these viruses, infectious molecular clones of SIVagm9063 and SIVagm155 have been previously described (22, 30). SIVagm155-4 was cloned from a PBMC isolate from AGM155 that had been passaged extensively in rhesus PBMC and thus is not entirely representative of the isolate from lymphoid tissues used in the present study. These viruses cluster within the vervet lineage of SIVagm, sharing 88, 83, and 80% identity, respectively, in Gag, Pol, and Env sequences. SIVagm9063 was isolated after in vivo passage of SIVagm90 in pigtailed macaque PT63 and, therefore, was presumed to have diverged genetically from the parental virus. To evaluate the degree of divergence of these two viruses, envelope sequences of SIVagm90 were cloned following RT-PCR amplification from the virus stock used for macaque inoculation. Envelopes derived from the SIVagm90 stock were closely related to one another (95% identity) and slightly more divergent from SIVagm9063 (90% identity) but significantly closer genetically than to SIVagm155. As shown in Fig. 1, variable residues were clustered in defined variable regions. The majority of potential N-linked glycosylation sites were conserved (n = 18), and all cysteine residues were conserved among the three strains. Distinct variable regions were observed with insertion/deletion polymorphisms in the V1 region. The cysteine loop that is analogous to the HIV-1 V3 loop (residues 274 to 307) was generally well conserved, consistent with previous observations on this region of SIVagm and SIVsm. However, a region of hypervariability was observed immediately C-terminal to this loop. Variable regions (V4 and V5) flanked the previous defined and highly conserved CD4-binding domain. The envelope clones of SIVagm90 and SIVagm9063 encoded full-length transmembrane (TM) proteins, whereas the TM protein of the molecularly cloned SIVagm155-4 was prematurely truncated in the cytoplasmic tail region. SIVagm155-4 was derived from a culture that had been passaged extensively in rhesus macaque PBMC, a procedure often associated with tissue culture adaptation. Macaques in the present study were inoculated with a virus isolated from tissues of AGM155 by short-term culture with a PT macaque PBMC isolate that encoded a full-length TM glycoprotein (data not shown).
Variable plasma viremia among SIVagm-infected macaques. Following intravenous inoculation, the macaques were monitored sequentially by virus isolation from PBMC, flow cytometric analysis of CD4+ and CD8+ T cells and B cells in blood, Western blot analysis of plasma for antibody responses, SIV-specific in situ hybridization of lymph node biopsy specimens, and plasma viral RNA loads. All of the animals became infected and seroconverted, as shown by Western blot analysis (data not shown). As shown in Table 1, infectious virus was successfully isolated from PBMC of each of the inoculated macaques during the early phases of infection. Virus was also recovered from lymph node mononuclear cells of each macaque at 1, 2, 4, and 8 weeks postinoculation (data not shown). Virus isolation from PBMC became less consistent at later time points in some of the animals (PT9636, PT9665, PT9657, PT9669, and PT7119), suggestive of low viral loads in these animals. This was particularly obvious in macaques inoculated with SIVagm155.
Plasma viremia was monitored sequentially throughout the course of infection by using real-time RT-PCR with SIVagmVer-specific primers and probe (19). As demonstrated in Fig. 2, high levels of primary plasma viremia were achieved in all animals by 1 week after inoculation (ranging from 107 to 108/ml of plasma), regardless of virus inoculum. Viral RNA loads in plasma declined sharply in all of the animals to varying degrees, reaching a semi-steady-state plateau (or setpoint) by approximately 8 to 12 weeks postinfection. The plateau levels varied widely between animals from 102 to 106 per ml, with the widest range among animals inoculated with SIVagm90. The plasma viral load setpoint was consistently high in SIVagm9063-inoculated macaques and consistently lower in SIVagm155-inoculated macaques. Four animals demonstrated good control of viremia to levels generally less than 1,000 copies/ml; these macaques included one inoculated with SIVagm90 and all three inoculated with SIVagm155. This was in complete agreement with the general difficulty in isolation of virus from PBMC (Table 1) of these animals. In contrast, virus isolation was consistently successful from animals with high plateau levels of plasma viremia.
Lymph node biopsy specimens sampled at 1, 2, and 4 weeks postinoculation were evaluated by SIVagm-specific in situ hybridization. The numbers of SIV-expressing cells observed in lymph node biopsy specimens was expressed as the average of the number of positive cells in 10 high power fields (40x objective), and the kinetics of virus expression in each of the animals is shown graphically in the top of Fig. 3. Large numbers of SIV-expressing cells were observed in 1-week biopsy specimens, concurrent with the peak of plasma viremia, and the numbers of SIV-positive cells declined by 2 weeks, with evidence of diffuse staining of germinal centers of follicles by 4 weeks postinoculation (see bottom of Fig. 3). As shown, peak virus expression was generally observed at 1 week postinfection (p.i.), coincident with peak plasma viremia, and subsequently declined to low but still detectable numbers of infected cells by 4 weeks p.i. Virus expression was extremely rare in lymph nodes biopsied from animals inoculated with SIVagm155. Thus, virus-expressing cells were only observed in lymph nodes sampled from this group of animals at 1 week and one from 2 weeks. The number of SIV-expressing cells was also lower than in SIVagm90- and SIVagm9063-infected macaques, and trapping of virus in germinal centers was not evident.
SIVagm90 infection results in blood CD4+-T-cell depletion. Lymphocyte subsets in the blood were monitored sequentially throughout infection. As shown in Fig. 4 and 5, a decline in CD4+ and CD8+ T cells and B cells was observed in all animals coincident with the peak of plasma viremia (i.e., 7 to 10 days p.i.). These lymphocyte subsets generally rebounded to near preinoculation values in the majority of animals. However, progressive CD4 depletion leading to CD4+-T-cell numbers of <100 CD4+ T cells/μl by approximately 6 months p.i. was observed in two of SIVagm90- and three SIVagm9063-inoculated PTs, respectively. Less-pronounced CD4 depletion was observed in the other animals in these two cohorts. In contrast, CD4+ T cells in the blood of SIVagm155-inoculated PTs remained relatively stable (Fig. 5). However, even in these three animals, the levels CD4+ T cells remained significantly lower than preinoculation values, with a slower trend toward loss of CD4+ T cells over time from inoculation (Fig. 4). CD8+-T-cell and B cell numbers also declined but never to the same degree as observed in the CD4+-T-cell subset. Some animals showed transient increases in B cell numbers (PT9657 and PT9669) or CD8 numbers (PT9668). Immunohistochemical analysis of lymph nodes, spleens, and gastrointestinal lymphoid tissues collected terminally in macaques with peripheral CD4-T-cell depletion confirmed a generalized depletion of CD4 T cells in lymphoid tissues (data not shown).
SIVagm90 and SIVagm9063 induce AIDS in PT macaques. Macaques were euthanized if they lost >20% body weight or had persistent diarrhea or other clinical symptoms that were not responsive to supportive and antibiotic therapy. As shown in Table 2, three animals inoculated with SIVagm90 and three animals inoculated with SIVagm9063 were euthanized over the first 2 years following inoculation. These animals included all the animals with severe CD4 depletion as well as one other (PT9636) that died due to thrombosis of the pulmonary artery, a complication we have observed previously in SIV-infected PT macaques (24). As detailed in Table 2, many of these animals had pathological evidence of severe enteritis with blunting of villus tips, lymphoid depletion in gastrointestinal-associated lymphoid tissues, edema, and crypt abscessation, although the etiologic agents were not identified. Three of the animals had bacterial infections, fulminant necrotizing bacterial pancreatitis and peritonitis (PT9529), bacterial pneumonia (PT9523), and disseminated lymphadenitis and enteritis due to Mycobacterium avium complex infection. Other AIDS-related pathological findings included membranoproliferative glomerulonephritis and pulmonary thrombosis. In situ hybridization for SIV expression in tissues collected terminally from these animals demonstrated a range in virus loads among animals, with the highest levels observed in PT9414 and PT9515 (data not shown). In contrast, all of the macaques inoculated with SIVagm155 remain clinically healthy at 140 weeks postinoculation.
Inverse correlation between plasma viral setpoint and CD4+-T-cell depletion. Since viral load and outcome varied greatly among these PT macaques, the correlation between the plasma viral setpoint and disease outcome was evaluated by using the Pearson correlation coefficient. We evaluated the correlation between the viral setpoint and various measures of disease outcomes including survival, slope of CD4+-T-cell loss, and the percentage of CD4 cells by 6 months postinoculation compared to preinoculation. Plasma viral load at setpoint was defined operationally as the viral RNA load at 12 weeks postinoculation. The percent CD4 loss by 6 months of infection correlated with survival (r = 0.782, P < 0.0001) and with the slope of CD4 loss over the 140 weeks of observation (r = 0.768, P = 0.0007), and therefore, since this has greater value in evaluating disease progression during infection, we used the percentage of CD4 cells remaining at 6 months as a general measure of disease progression. As shown in Fig. 6, a statistically significant inverse correlation was observed between the log10 plasma viral load at the setpoint with the percent loss of CD4+ T cells at 6 weeks postinfection (r = –0.951, P < 0.0001). Thus, the higher the plasma viral load, the greater the CD4+-T-cell depletion observed. The viral setpoint also correlated inversely with survival (r = –0.742, P < 0.01).
Cursory inspection of virus load and CD4 loss in these animals suggested that SIVagm155 was less pathogenic than either SIVagm90 or SIVagm9063. We therefore used Student's t test to compare peak viral load (log10), setpoint viral load, and the percentage of CD4 T cells at 6 months postinoculation among the three groups of animals. As shown in Table 3, the peak viral load did not differ significantly among the three groups. In addition, setpoint viremia and CD4 loss did not differ significantly between SIVagm90- and SIVagm9063-inoculated macaques (P = 0.739 and P = 0.402, respectively). However, setpoint viremia was significantly higher in SIVagm90/9063-inoculated macaques than in SIVagm155-inoculated macaques (P = 0.0006), and these latter animals also maintained significantly higher levels of CD4+ T cells than SIVagm90/9063-infected PTs (P = 0.004). However, despite reduced virulence of SIVagm155 and apparent clinical health, these animals do not appear to be immunologically normal. They all exhibit some degree of lymphadenopathy, and their CD4+-T-cell numbers are reduced compared to preinoculation values, suggesting that, even at low virus loads, SIVagm infection has pathological consequences in PT macaques.
DISCUSSION
The present study confirms the pathogenicity of SIVagm90 and a macaque-passaged derivative (SIVagm9063) in PT macaques. Infection of PT macaques with either of these virus isolates resulted in the progressive loss of circulating CD4+ T lymphocytes, severe lymphoid depletion, and the development of an AIDS-like syndrome. Macaque passage of this virus was not required for pathogenicity, although the virus was not uniformly pathogenic in this small cohort of macaques. We therefore conclude that this natural virus isolate is fully competent to induce AIDS in macaques without genetic and biologic selection in this foreign host species. Since SIVagm90 was isolated in macaque PBMC, we cannot rule out the possibility that growth in macaque PBMC results in some selection of already-existing members of the viral quasispecies. This study also clearly demonstrates that not all natural isolates of SIVagm are equally pathogenic; infection of PT macaques with the related SIVagm155 was rapidly controlled and did not induce AIDS in a period of observation of 140 weeks. Pathogenic and nonpathogenic SIVagm infection of macaques could not be distinguished by the levels of primary viremia, which was robust in all inoculated animals, but disease progression was associated with persistent plasma viremia.
Pathogenic infection of macaques with SIVagm appeared to be similar to that of SIVsm/mac infection of macaques, with perhaps a slower rate of disease progression. These animals developed lymphadenopathy, progressive CD4+-T-cell decline with the development of terminal opportunistic infections and showed a similar disease course and kinetics of primary and setpoint viremia. Critically, SIVagm was similar to SIVmac/sm infection in the demonstration of an inverse correlation of virus load in plasma with disease course. Thus, the mechanisms of AIDS induction are likely to be common between these pathogenic virus infections. Although there are clearly aspects of SIVagm infection that have not yet been explored, such as the generation of cytotoxic T cell and neutralization escape mutants or effects on CCR5-expressing memory CD4 T cells in mucosal sites, we conclude that SIVagm infection of PT macaques is a valid animal model to explore pathogenic mechanisms of human AIDS.
While SIVagm-PT macaque model is not a novel model for AIDS, the ability to compare and contrast pathogenic (macaque) and nonpathogenic (AGM) infection in different host species with a common virus strain may give unique insights into the pathogenesis of AIDS in humans. Despite years of study, it is apparent that the mechanisms underlying immunodeficiency in HIV and SIV infection are poorly understood. The correlation between plasma viremia and rapidity of disease progression in pathogenic SIV and HIV infections suggests that much of the CD4 depletion that occurs in AIDS is the direct result of virus-induced cell death. Initially, AIDS was assumed to be due to direct killing of CD4+ T cells, an obvious conclusion based upon the fact that HIV and SIV use CD4 as their cellular receptor. However, AIDS can occur in both humans and macaques in the face of incomplete CD4+-T-cell depletion (26), suggesting qualitative dysfunction of CD4+ T cells or specific subset depletion. Indeed, earlier studies of SIVmac infection showed early selective depletion of CCR5+, memory CD4+ T cells in the gastrointestinal tract and other mucosal systems (60). A recent study of SIVsm-infected macaques revealed a similar early depletion of CCR5+ memory cells in the peripheral blood; this contrasted with depletion of CXCR4+ naive cells in simian human immunodeficiency virus infection (41). These data collectively suggest that much of the early immune dysfunction in SIV and HIV infection occurs by depletion of a subset of CD4+-T-cell populations such that the host is never able to gain advantage over the virus infection. SIV-infected macaques survive for as long as 2 years after infection with very gradual deterioration of the immune system, suggesting a complex interplay between the virus and the immune system that involves early destruction of memory T cells by virus, followed by increased overall memory T cell turnover, thymic dysfunction, and limitations in peripheral T cell regeneration (14).
The comparative study of pathogenic and nonpathogenic (natural) models of SIV infection is likely to give additional insight into the mechanisms of AIDS. Since the correlation between viral replication levels and disease have been a constant theme in SIV infection of macaques and HIV infection of humans, the initial assumption regarding the lack of disease in natural host species was that it resulted from strict containment of viral replication, analogous to what is observed with long-term nonprogressors of HIV infection (9). However, this is clearly not the case. Naturally infected AGM (8, 19, 27), sooty mangabeys (10, 11, 32, 52), and mandrills (42, 58) appear to have significant, ongoing viral replication, similar to levels during the asymptomatic phase of pathogenic infection in macaques and humans. Genetic evolution of SIV in both AGM and sooty mangabeys also suggests ongoing, persistent viral replication (10, 39, 46). Thus, natural models of SIV are not analogous in virologic terms to long-term nonprogressors of HIV infection. Studies with this virus in AGM demonstrate robust virus replication (unpublished data) comparable to levels of viremia observed in naturally infected AGM. This present study demonstrates that the natural SIVagm90 isolate would be appropriate for comparative studies of SIVagm in PT and AGM. We believe it is critical to use a common virus in these two species for comparative purposes to allow the use of common assays and reagents. The use of SIVagm90 in AGM would be appropriate for modeling natural infection of AGM, since it is not adapted to macaques, and will be the focus of future studies.
ACKNOWLEDGMENTS
We thank C. Erb, S. Whitted, and R. Goeken of LMM, NIAID, for technical assistance and M. A. Robinson and T. Myers of RTB, NIAID, for assistance on statistical analysis.
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