Comparison of Simian Immunodeficiency Virus SIVagm
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病菌学杂志 2006年第10期
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Bethesda, Maryland 20892
Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, Louisiana 70433
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Research East 113, 41 Avenue Louis Pasteur, Boston, Massachusetts 02115
ABSTRACT
The simian immunodeficiency viruses (SIV) naturally infect a wide range of African primates, including African green monkeys (AGM). Despite moderate to high levels of plasma viremia in naturally infected AGM, infection is not associated with immunodeficiency. We recently reported that SIVagmVer90 isolated from a naturally infected vervet AGM induced AIDS following experimental inoculation of pigtailed macaques. The goal of the present study was to evaluate the replication of this isolate in two species of AGM, sabaeus monkeys (Chlorocebus sabaeus) and vervets (C. pygerythrus). Inoculation of sabaeus AGM with SIVagmVer90 resulted in low and variable primary and set-point viremia (<102 to 104 copies/ml). In contrast, inoculation of vervet AGM with either SIVagmVer90 or blood from a naturally infected vervet (Ver1) resulted in high primary viremia and moderate plateau levels, similar to the range seen in naturally infected vervets from this cohort. CD4+ T cells remained stable throughout infection, even in AGM with persistent high viremia. Despite the lack of measurable lymphadenopathy, infection was associated with an increased number of Ki-67+ T cells in lymph node biopsies, consistent with an early antiviral immune response. The preferential replication of SIVagmVer in vervet versus sabaeus AGM shows that it is critical to match AGM species and SIV strains for experimental models of natural SIV infection.
INTRODUCTION
The simian immunodeficiency viruses (SIV) are a diverse group of lentiviruses that infect a wide variety of African nonhuman primates, including sooty mangabey monkeys (SM) and African green monkeys (AGM). Molecular studies of SIV strains have demonstrated phylogenetic lineages that are specific for the primates of origin. For example, SIV lineages have been identified that are specific for SM (SIVsm) and AGM (SIVagm). The initial identification of distinct subtypes of SIVagm (SIVagmGri, -Ver, -Sab, and -Tan) specific to the four geographically separated AGM species, namely, grivet (7), vervet (1, 22, 45), sabaeus (21), and tantalus (18, 29) monkeys (Chlorocebus aethiops, C. pygerythrus, C. sabaeus, and C. tantalus, respectively), suggested that these viruses have coevolved with their primate host species over a long period of time (3, 29). Despite high seroprevalence rates in feral and captive populations, natural infections of African primates with their own SIV strains are not generally associated with immunodeficiency (16). While it is difficult to assess the effect of SIV infection in wild populations, large numbers of AGM and SM have been studied in captivity over long periods of time. The conclusion of these various studies is that SIV infections of natural host species are generally asymptomatic. CD4+ T-cell depletion has been observed in a few isolated cases, including a naturally infected mandrill (36), an African green monkey (44), and, most recently, a sooty mangabey (27), suggesting that in some circumstances these viruses may produce AIDS after long incubation periods. These studies suggest that African primates are not immune to the pathogenic effects of primate lentiviruses under specific circumstances. SIV strains are not inherently apathogenic, since experimental cross-species transmission to Asian macaque species results in an AIDS-like syndrome (17, 46). The mechanistic basis for the lack of disease in naturally infected species such as AGM may therefore provide insight into the pathogenic mechanism of AIDS and suggest novel therapeutic strategies.
An intriguing feature of natural infections of sooty mangabeys (SIVsm), AGM (SIVagm), and mandrills (SIVmnd) is the failure to develop AIDS in the face of persistent moderate to high viral loads (4, 11, 35, 37, 41). Experimental infections of mandrills with SIVmnd (33, 35) and of sabaeus AGM with SIVagm/Sab (6) resulted in high levels of primary viremia followed by partial containment of viremia. Therefore, the lack of disease in these SIV-infected African monkeys is clearly not due to suppression of viremia such as that observed in long-term nonprogressors among HIV-infected humans (5). Despite vigorous studies of both sooty mangabeys and African green monkeys, the mechanisms whereby these animals avoid AIDS are not clear (4, 13, 28, 39, 41). Therefore, experimental animal models that recapitulate the virologic and immunologic features of infection are essential. The goal of the present study was to establish an animal model using our previously characterized SIVagmVer90 isolate by investigating the viral replication of SIVagmVer90 in two species of AGM, vervets and the more readily available Caribbean sabaeus AGM.
MATERIALS AND METHODS
Animals and viruses. The naturally infected African green monkey (AGM90) that was the source of SIVagmVer90 was imported from Kenya in 1987 and phenotypically appeared to be a vervet monkey (11). The virus was isolated from the mesenteric lymph nodes of monkey AGM90 by coculture of viably frozen mononuclear cells with macaque peripheral blood mononuclear cells (PBMC). Viruses were titrated for infectivity in the susceptible cell line CEMss. Six sabaeus AGM and four vervet AGM were inoculated intravenously with 1,000 50% tissue culture infective doses (TCID50) of SIVagmVer90. Animals were maintained in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals under an NIAID-approved animal study protocol (32) and were housed in a biosafety level 2 facility using biosafety level 3 practices. The vervet AGM utilized for this study were imported from Tanzania and screened for SIV infection by Western blotting, radioimmunoprecipitation, virus isolation from PBMC, and measurement of plasma viral RNA (vRNA) loads. One seropositive vervet AGM (Ver1) was used as a source of SIVagm-infected blood for transfusion of 2 ml of heparinized blood into four seronegative vervets. A limiting 10-fold-dilution coculture of PBMC isolated from AG1 blood collected at the time of transfusion was performed with CEMss cells. Virus isolation required 106 PBMC, and thus this inoculum represented approximately 50 TCID.
Genetic characterization of vervet AGM by cytochrome b gene sequencing. In order to confirm the species of vervet AGM, DNAs were extracted from 300 μl of whole blood from four SIV-seronegative monkeys from the Tanzanian cohort (Ver7, Ver9, Ver13, and Ver23). A QIAamp blood kit (QIAGEN, Valencia, CA) was used to extract the DNAs, which were eluted into 60 μl of H2O and stored at –20°C. A 267-bp region of the cytochrome b gene, representing bases 14841 to 15149 of the human mitochondrial genome (2), was amplified using primers L14841 and H15149 (20). One microgram of DNA was used in a 100-μl PCR mix for PCR in a Perkin-Elmer PE 9700 thermocycler. The PCR conditions were as follows: 2 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C, with a final extension of 5 min at 72°C. Following purification with a QIAquick PCR purification kit (QIAGEN), the PCR products were cycle sequenced and analyzed on an ABI 377 automated DNA sequencer. Cytochrome b gene sequences from different species of AGM were aligned with that from a grivet (GenBank accession no. AY863426) and with sequences from other African nonhuman primates retrieved from GenBank. Sequences were aligned using ClustalW 1.8. Unrooted maximum parsimony trees were inferred using PAUP, version 4.0b8, and were uniformly weighted and unordered.
Virus isolation. Viruses were isolated from PBMC of experimentally infected AGM. Ficoll-separated PBMC were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, stimulated with 5 μg per ml phytohemagglutinin, and cultivated for 3 days in the presence of 20 units per ml of interleukin-2. Subsequently, 5 x 106 PBMC were cocultivated with CEMss cells for 6 weeks, with biweekly feeding, and the culture supernatant was monitored for reverse transcriptase (RT) activity as previously described (10).
Plasma vRNA assay. A real-time RT-PCR assay for quantitation of viral RNA in plasma was performed as previously described (11), using methodology based on the 7700 sequence detection system (Applied Biosystems, Foster City, CA) that was used previously for SIVsm/mac-specific real-time PCR (43). Briefly, forward and reverse primers to amplify a 122-bp fragment and an internal fluorogenic probe were generated based on the SIVagm155 sequence (GenBank accession no. M29975), as follows: AgmF, 5'-GTC CAG TCT CAG CAT TTA CTT G-3' (7981); AgmR, 5'-CGG GCA TTG AGG TTT TTC AC-3' (nucleotide 8090); and probe, 5'-R-CAG ATG TTG AAG CTG ACC ATT TGG GQ-3' (nucleotide 8041), where R indicates a 6-carboxyfluorescein group and Q indicates a 6-carboxytetramethylrhodamine group conjugated through a linker arm nucleotide linkage. Previous studies have shown that this primer/probe set amplifies divergent SIVagmVer isolates (11). Sequence analysis of env clones from Ver1 showed that sequences in the areas of both primers were conserved. There is one mismatch of Ver1 env with the probe sequence which is shared with SIVagmVer90. The high-performance liquid chromatography-purified probe was obtained from Applied Biosystems/Perkin-Elmer (Foster City, CA). Plasma samples for analysis were collected by using EDTA as an anticoagulant and were stored in a –80°C freezer until analysis. Plasma viral RNAs were isolated using a QIAmp viral RNA kit (QIAGEN, Valencia, CA). Sense RNA transcribed using T7 polymerase from an EcoRI-linearized plasmid created from a 1.9-kb fragment (HindIII/HincII) of the envelope of pSIVagm9063-2 cloned into the pTRI-10 [poly(A)] plasmid vector was used as a standard for the assay. RT reactions were performed in 96-well plates, and the mixtures contained identical concentrations of the following components in RNase-free water: random hexamers (2.5 μM; Promega, Madison, WI), 5.0 mM MgCl2, a 1.0 mM concentration of each deoxynucleoside triphosphate, 5.0 mM dithiothreitol, and 20 U Superscript RT.
SIV-specific antibodies. Serology for antibodies to SIVagm was performed by Western blot analysis, as previously described (17). Briefly, the virus was pelleted from the cell-free supernatant of SIVagmVer90-infected CEMss cells, and virus lysates were electrophoresed in a polyacrylamide gel, transferred to nitrocellulose, and used as an antigen to react with macaque plasma. SIV antibodies were detected by using an anti-human immunoglobulin G antibody conjugated with alkaline phosphatase (Amersham, Piscataway, N.J.). Bands were detected upon the addition of the BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) phosphatase substrate system (KPL Laboratories, Gaithersburg, MD). The serologic status of the wild-caught vervets was confirmed by radioimmunoprecipitation as previously described (14), using CEMss cells infected for a short term with SIVagmVer90 as the source for cell lysates.
Absolute CD4+ T-cell counts. CD4+ T cells were analyzed sequentially throughout infection by flow cytometry. Briefly, EDTA-anticoagulated 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 a fluorochrome. The monoclonal antibodies used to identify CD4+ T-cell subsets were L200-allophycocyanin (BD Biosciences, San Jose, CA) for vervets and OKT4a-fluorescein isothiocyanate (Ortho Diagnostic Systems, Raritan, N.J.) for sabaeus monkeys. Following staining, erythrocytes were lysed, and leukocytes were fixed in 1% paraformaldehyde and analyzed using either a Becton Dickinson FACSVantage SE DiVa cell sorter, for specimens from vervets, or a Coulter Epics 753 cell sorter, for specimens from sabaeus monkeys. To determine the absolute CD4+ T-cell counts, the percentages of CD4+ T lymphocytes were multiplied by the absolute lymphocyte counts obtained using the ADVIA 120 hematology system (Bayer, Tarrytown, NY).
SIV-specific in situ hybridization. Nonradioactive in situ hybridization (ISH) for SIV expression was performed on formalin-fixed, paraffin-embedded lymph nodes, utilizing sense or antisense digoxigenin-labeled riboprobes that spanned the entire SIVagm9063-2 genome, as previously described (17). Briefly, sections were deparaffinized and rehydrated with water, pretreated with 0.2 N HCl and proteinase K, and then hybridized overnight at 51°C with either the antisense or the sense riboprobe. The riboprobe consisted of a mixture of probes conjugated with digoxigenin-UTP (Lofstrand Labs, Ltd., Gaithersburg, Md.). The hybridized sections were washed in standard posthybridization buffers and RNase A solutions (RNase A [Sigma] and RNase T1 [Roche Molecular Biochemicals, Indianapolis, Ind.]). The sections were blocked with 3% normal sheep and horse sera 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, incubated with NBT/BCIP (Vector Laboratories, Ltd., Burlingame, Calif.) for 10 h, and visualized with a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, N.Y.).
Immunohistochemistry. Ki-67 expression was assessed in sequential formalin-fixed, paraffin-embedded lymph node samples by immunohistochemical staining as previously described (19). Samples were incubated with Ki-67 (DAKO) and goat anti-rabbit immunoglobulin G-Alexa fluor 488 (Invitrogen). The stained sections were rinsed, counterstained with hematoxylin, covered, and photographed with a Zeiss Axiophot microscope equipped with a Cool Snap digital camera (Biovision). Sections were analyzed by segmenting (counting) the total number of SIV-positive cells by ISH (IP Lab software) from three representative fields of view of the paracortical region of each section. The total number of Ki-67+ cells per region of interest was calculated and imported into Prism 4, and the means and standard errors of the means (SEM) were graphed.
Sequence analysis. PCR was used to amplify a 370-bp fragment of the transmembrane glycoprotein-encoding region of envelope from 500 ng of genomic DNA extracted from CEMss cell isolates of SIVagm from 12 SIV-seropositive AGM from the Tanzanian cohort. Primers for amplification were designed based on an alignment of published SIVagm/Ver isolates and had the following sequences: forward primer, 5'-CCT CAA TGC CCG CGT CAC AGC-3'; and reverse primer, 5'-CTC CTG CGT TGT CTG GCT GTC-3'. The resulting products were purified by electrophoresis in a 2% agarose gel, extracted from the gel using a QIAquick PCR purification kit (QIAGEN), and sequenced directly using the PCR primers and a Terminator cycle sequencing kit for fluorescence-based sequencing (Applied Biosystems, Warrington, United Kingdom). Phylogenetic analysis was performed using neighbor joining calculated from a Kimura two-parameter distance matrix (MacVector).
Statistical methods. The differences in viral loads and CD4+ T-cell numbers between different groups of monkeys at various time points were determined by the Mann-Whitney test using GraphPad Prism, version 4, software (San Diego, CA).
RESULTS
Selection and characterization of study animals. Six Caribbean green monkeys were imported from St. Kitts and screened serologically for SIV and simian T-cell leukemia virus type 1 (STLV-1) infection. As expected, all of these animals were seronegative for SIVagm. These animals evolved from a founder population imported from West Africa over 300 years ago and are presumed to be of the sabaeus species based upon their origin and phenotypic appearance. As shown in Fig. 1, sequence analysis of cytochrome b genes from Caribbean AGM are also consistent with their classification as sabaeus AGM (I. Pandrea, C. Apetrei, J. Dufour, N. Dillon, J. Barbercheck, M. Maetzger, B. Jacquelin, R. Bohm, P. A. Marx, F. Barre-Sinoussi, V. Hirsch, M. C. Muller-Trutwin, A. A. Lackner, and R. S. Veazey, submitted for publication). Cytochrome b gene analysis was not performed on the sabaeus monkeys used in the present study since they were animals obtained from the same source as the Caribbean sabaeus AGM used for Fig. 1.
Twenty-four wild-caught AGM were imported from Tanzania, a part of the known geographical habitat of vervets. Phylogenetic analysis of cytochrome b genes from four representative animals from this cohort confirmed that they were genetically related to other AGM but formed a phylogenetic cluster distinct from sabaeus, tantalus, and grivet monkeys (Fig. 1), consistent with their classification as vervets. Western blot analysis of plasma samples identified 12 of the vervets as SIV seropositive, and viral RNA was detected in 11 of these by a vervet-specific real-time RT-PCR assay. Viral RNA was not detected in the plasma of one of the vervets (Ver15), presumably due to a mismatch in the primer/probe sequences. Plasma vRNA levels were consistent in individual animals upon six repeated monthly assays, with mean values for the individuals ranging from 3,400 to 217,000 copies/ml of plasma (Table 1). A 370-nucleotide fragment of the transmembrane portion of env was amplified by PCR from DNAs extracted from viruses isolated from PBMC of the seropositive vervets. This region spans the extracellular variable region of the transmembrane protein and could be readily amplified using highly conserved primers. As shown in Fig. 2, each of the envelope sequences was distinct, but the sequences clustered phylogenetically with previously characterized SIVagm isolates from vervet monkeys (SIVagmVer90 and SIVagmVer155) and were distinct from representative SIVagm isolates from the other three species of AGM, i.e., grivet, sabaeus, and tantalus monkeys. Eight of the 12 seronegative vervets were chosen for subsequent experimental inoculation with SIVagmVer, and one vervet (Ver1) with high viremia was chosen as a source of blood for the inoculum.
Inoculation of sabaeus and vervet AGM with SIVagmVer. Four vervets (Ver20, Ver21, Ver22, and Ver24) and six sabaeus monkeys (Sab951, Sab954, Sab955, Sab964, Sab005, and Sab038) were inoculated with the same dose and stock of cryopreserved SIVagmVer90 used in our previous study of pig-tailed (PT) macaques (12). An additional four vervets (Ver2, Ver8, Ver14, and Ver19) were inoculated with 1 ml of EDTA-anticoagulated blood from the naturally infected vervet, Ver1. All of the animals were inoculated intravenously and monitored sequentially by virus isolation from PBMC and lymph node biopsies, flow cytometric analysis of peripheral blood CD4+ T lymphocytes, plasma viral RNA levels, SIV-specific ISH of lymph nodes, and SIV serology by Western blot assay.
All animals became infected, as evidenced by the isolation of infectious virus from PBMC or lymph nodes by cocultivation with CEMss cells following stimulation with phytohemagglutinin and interleukin-2. Virus isolation from vervet PBMC was highly reproducible at sequential time points throughout infection. In contrast, virus isolation from sabaeus monkeys inoculated with SIVagmVer90 was inconsistent (Table 2). Virus was only occasionally isolated from PBMC samples of two of the sabaeus AGM (Sab951 and Sab954). For example, virus was only isolated from lymph node biopsies collected from Sab951 at 1 and 4 weeks postinoculation and not from PBMC at any other time points. These data suggested more robust replication of SIVagmVer viruses in vervet monkeys than in sabaeus monkeys.
High viremia in vervets but not sabaeus monkeys inoculated with SIVagmVer. For a more quantitative assessment of virus replication, plasma viral RNA was measured using a real time RT-PCR assay developed specifically for vervet isolates of SIVagm (11). As shown in Fig. 3A and B, primary viremia was consistently high in vervets inoculated with either SIVagmVer90 or SIVagmVer1 (106 to 108 copies/ml of plasma). Peak viremia was slightly higher for vervets that received blood from Ver1, and the peak was slightly delayed compared to that for vervets inoculated with SIVagmVer90 (10 days versus 7 days). This delay in peak viremia in vervet AGM inoculated with AG1 blood may have been due to the difference in the sizes of the inocula (50 versus 1,000 TCID). Primary infection of vervets was followed by an abrupt decline in plasma viremia by 4 weeks postinoculation in all animals and then the establishment of a stable plateau level of 104 to 106 copies/ml. Therefore, the replication kinetics of SIVagmVer90 and SIVagmVer1 were similar in vervet monkeys.
A much wider range in primary viremia was observed for the sabaeus monkeys (Fig. 3C and D), ranging from undetectable (Sab951) to as high as 106/ml (Sab964). The peak of primary viremia was also delayed, to 2 to 3 weeks postinoculation, compared to 1 week postinoculation for the vervets. The two sabaeus monkeys (Sab955 and Sab964) with the most consistent isolation of SIV from PBMC (Table 2) also had the highest levels of primary viremia. An abrupt decline in plasma viremia was observed in all sabaeus monkeys by 8 to 12 weeks postinoculation, and all of the animals except for Sab951 maintained detectable levels of plasma vRNA throughout the 100-week period of observation. As observed during primary infection, the range in chronic viremia was very broad and dependent on the individual, ranging from undetectable (<102) to 106 copies/ml of plasma.
A comparison of sequential mean viral RNA levels (Fig. 3D) among AGM inoculated with SIVagmVer clearly demonstrated that SIVagmVer90 replication was less robust in sabaeus monkeys than in vervets, suggesting a specific host adaptation. Primary viremia was significantly lower (P < 0.01) in sabaeus monkeys than in vervets infected with SIVagmVer90, with no overlap between values. Set-point viremia was also significantly lower in the sabaeus monkeys (P < 0.01), ranging from <100 to 5,700 copies/ml, than in the vervet AGM (8,000 to 300,000 copies/ml). In contrast, the viral replication kinetics of SIVagmVer90 and SIVagmVer1 in vervets were similar. The peak of primary viremia in vervets inoculated with SIVagmVer1 was slightly delayed relative to that in vervets inoculated with SIVagmVer90 (Fig. 3D). However, there was no significant difference in viral loads in the postacute period of infection in vervets inoculated with either SIVagmVer90 or Ver1 blood (P = 0.32).
Stable blood CD4+ T-lymphocyte numbers in SIVagm-infected AGM. CD4+ T lymphocytes in the peripheral blood of AGM were monitored over a period of 90 weeks following inoculation (Fig. 4). A transient decline in CD4+ T cells was observed during primary infection in a subset of the animals. CD4+ T cells in peripheral blood remained stable throughout the entire period of observation, with no differences observed between the different species inoculated. All of the AGM remained asymptomatic, although one vervet (Ver21) was euthanized at 70 weeks postinoculation due to non-AIDS related causes (self-mutilation). The lack of peripheral CD4+ T-cell depletion in both vervets and sabaeus monkeys in the present study contrasted with the marked depletion we observed previously in PT macaques inoculated with SIVagmVer90 (12).
Robust envelope-specific antibody responses in AGM. SIV-specific antibody responses of vervet and sabaeus AGM inoculated with SIVagmVer90 were monitored by Western blotting using SIVagmVer90 as the viral antigen. As shown in Fig. 5, all but one of the AGM (Sab951) developed SIV-specific antibody responses. This particular animal exhibited the most inconsistent virus isolation from PBMC and lymph nodes, and viral RNA was only detected at one time point, all of which are consistent with a transient or subclinical infection. All other animals responded with robust levels of envelope-specific antibody, but with much weaker responses to Gag or other viral antigens. The predominance of envelope-specific antibody responses has been observed previously by us and others in naturally infected AGM (11, 31), and its significance is not clear in terms of its relationship to the lack of disease in AGM.
SIV-specific ISH and immunohistochemical analysis of lymph nodes. SIV-specific ISH was performed on sequential lymph node biopsies sampled at 1, 2, and 4 weeks postinoculation (Fig. 6). A comparison of SIV expression in lymph nodes in vervets and sabaeus monkeys inoculated with SIVagmVer90 revealed robust infection in lymphoid tissues of the vervets. A similar level of expression was observed in the vervets inoculated with SIVagmVer1 (data not shown). Representative fields are shown in Fig. 6B to demonstrate the large number of SIV-expressing cells in lymph node biopsies collected at 1 week from vervets inoculated with SIVagmVer90 and the subsequent decrease by 2 weeks. This high level of expression contrasted with the low expression of SIV in the lymph nodes of sabaeus monkeys inoculated with SIVagmVer90. SIV-expressing cells were only observed in one lymph node biopsy collected from H964 at 1 week postinoculation (peak viremia). Thus, the expression of SIV in lymph nodes reflected the differences in primary viremia observed between vervets and sabaeus monkeys inoculated with a common SIVagmVer90 strain.
The lymph nodes of both vervets and sabaeus AGM remained constant in size throughout infection, without evidence of paracortical expansion. This contrasts with the marked lymphadenopathy observed in SIVagmVer-inoculated PT macaques (12, 16). We focused on analyzing the vervets since the reduced viral replication observed in the sabaeus monkeys suggested that they might not be reflective of natural infection. To assess the extent of immune activation occurring with lymph nodes during the first month of infection, Ki-67 expression was assessed in formalin-fixed lymph nodes collected preinoculation and 1, 2, and 4 weeks after infection from the SIVagmVer90-inoculated vervets. As shown graphically in Fig. 7A, the number of Ki-67+ cells in the paracortical areas of the lymph nodes increased significantly following infection (P < 0.01), reaching peak levels by 2 weeks and subsiding by 4 weeks. As evident in a representative section in Fig. 7B, preinoculation lymph nodes were relatively quiescent, with small numbers of Ki-67+ cells in the paracortical regions (P) and rare germinal centers (GC). By 2 weeks postinoculation, developing germinal centers stained intensely for Ki-67, consistent with the expansion of B lymphocytes (Fig. 7B, bottom panel). Confocal analysis showed that Ki-67+ cells in the paracortex (data not shown) represent a mixture of CD4+ and CD8+ T cells, with a predominance of CD8+ T cells.
DISCUSSION
This study extends our previous evaluation of a primary SIVagmVer isolate in PT macaques, a pathogenic model (12) of the natural AGM host for this virus. The levels of chronic viremia established in vervet AGM in the present study were similar to the levels observed in the naturally infected cohort of vervets and to previous reports of natural SIVagm infection in AGM (4, 11). In addition, the pattern of primary and chronic viremia observed in vervet monkeys infected with SIVagmVer90 was similar to that in previous reports of sabaeus monkeys experimentally inoculated with SIVagmSab (34). Virus replication levels following infection with SIVagmVer90 and SIVagmVer1 (blood inoculum) were similar in vervets, suggesting that SIVagmVer90 has not been significantly modified in biologic terms by passage in macaque PBMC. However, the magnitude of viremia of SIVagmVer90 was dependent on the particular species of AGM used for experimental infection. Despite the fact that the sabaeus and vervet species exhibit only minor differences in genes that are critical for SIV infection, such as the CD4 and CCR5 genes (8, 30), SIVagmVer replicated significantly more efficiently in vervets than in sabaeus monkeys. Although viral replication of SIVagmVer in vervets and sabaeus monkeys differed, the clinical outcomes were similar. All animals remained asymptomatic for 90 weeks following infection. Despite different levels of persistent viremia among the AGM, peripheral blood CD4+ T-cell levels were remarkably stable.
The present study confirms recent observations of significant differences in the replication of specific SIVagm isolates in different species of AGM. A previous study showed that SIVagmSab replicated efficiently in both sabaeus and vervet AGM (34) and that SIVagmVer replicated inefficiently in both sabaeus and vervet AGM, suggesting virus-specific replication. This contrasts with the results of our study, where we observed robust replication of SIVagmVer90 and SIVagmVer1 in vervet AGM but inefficient replication of these viruses in sabaeus AGM; thus, virus replication was dependent on the host species inoculated with SIVagmVer. Efficient replication was only observed in the homologous host. Although the previous study observed virus-specific replication differences, it is clear from these two studies that the virus and host species must be carefully considered and matched to the virus for natural history and virologic studies of SIVagm in vivo. We can only speculate on the host factors responsible for the host restriction of SIVagmVer in sabaeus monkeys. Since the restriction in viral replication occurs during primary infection, the mechanisms are likely to be related to innate immunity or host restriction factors, such as APOBEC3G (9, 15) or TRIM5alpha (23, 38, 42). Alternatively, factors involved in virus entry, such as CCR5 and CD4, may be responsible; this hypothesis seems unlikely since there are only minor differences in the sequences of CD4 and CCR5 (8, 25, 26, 30) between these two species.
Finally, the kinetics of early viremia in vervet monkeys inoculated with SIVagmVer was indistinguishable from that of viremia observed in PT macaques inoculated with SIVagmVer90 (12). This study confirms recent studies of SIVsm infections of sooty mangabeys and macaques that showed robust replication kinetics for both asymptomatic and pathogenic infections (40). Since viral replication was similarly robust in pathogenic and natural asymptomatic infections, it is likely that significant differences in host responses to SIV infection are responsible for divergent disease outcomes. Previous studies have suggested that the levels of virus-induced immune activation may be significantly lower in naturally infected host species than in hosts which develop AIDS (40, 41). In the present study, we demonstrated significant proliferation (as assessed by Ki-67 expression in lymph nodes) in SIVagmVer-inoculated vervet AGM. Such an increase in Ki-67+ cells is probably the result of a "normal" immune response that would be expected in any viral infection. This establishes that these animals are not inherently unresponsive to SIV infection and suggests that qualitative and dynamic differences in their reactions to infection compared to those in pathogenic macaque models may be responsible, in part, for their lack of disease. A side-by-side comparison with SIVagmVer-infected PT macaques will be necessary to evaluate whether this parameter differs in the two species. A preliminary analysis of immune activation in SIVagmVer-inoculated PT macaques showed similar kinetics of Ki-67-expressing cells in lymph nodes (data not shown). However, the numbers of Ki-67+ cells were always higher in PT macaques, even prior to SIV inoculation (data not shown). This finding is in agreement with comparative studies of sooty mangabeys and macaques infected with SIVsm, where greater numbers of Ki-67+ cells were observed in T-cell areas of macaque lymph nodes at 4 weeks postinfection (40).
Recent studies of AGM experimentally inoculated with SIVagm may shed some light on the potential mechanism for the lack of hyperimmune activation observed in AGM. Infection of sabaeus AGM with SIVagmSab was found to be associated with an early anti-inflammatory profile of gene expression, characterized by the induction of transforming growth factor 1 and FoxP3 and an increase in regulatory T cells (24); this profile differs from those observed for SIVmac infection of macaques and human AIDS, which are both associated with aberrant chronic T-cell activation. These data suggest that this early environment promotes the generation of regulatory T cells, which could dampen or prevent chronic hyperactivation. A direct comparison of AGM and macaques inoculated with a common virus will therefore be highly informative in determining the significance of immune activation in CD4+ T-cell-induced cell death and AIDS. Other potential mechanisms that will also be investigated include differences in cytopathic effects, CD4 regenerative ability, and disparity in target cell type or availability between the two hosts.
In summary, different species inoculated with a common virus (SIVagmVer) had the following distinct outcomes: (i) high viremia with disease in PT macaques, (ii) high viremia with no disease in vervet AGM, and (iii) moderate to low viremia with no disease in sabaeus AGM. SIVagmVer appears to be specifically adapted to vervet monkeys, in contrast to sabaeus AGM, with these species differing in terms of permissiveness for infection. In contrast, PT macaques and vervet AGM appear to be relatively equivalent in terms of their permissiveness for infection. However, these two species differ widely in terms of disease induction, with macaques developing AIDS (12) and vervets remaining healthy.
Preliminary studies suggest that vervets and macaques differ with respect to the extent of immune activation associated with infection, similar to observations for SIVsm infections of sooty mangabeys and macaques (40). Together, these data suggest that the level of immune activation may be a key determinant of disease progression. Experimental infections of vervet AGM and PT macaques with SIVagmVer90 will provide a valuable model system to explore differences in immunologic responses and target cells that differentiate asymptomatic natural infections from AIDS.
ACKNOWLEDGMENTS
We thank S. Whitted and R. Goeken of LMM, NIAID, and Cristian Apetrei of Tulane National Primate Research Center for technical assistance and Russell Byrum of Bioqual Inc. for conducting the animal studies.
This study was supported by the intramural program of the National Institute of Allergy and Infectious Diseases, NIH, and by NIH grants RO1 AI065335 (flow cytometric studies) and RO1 AI064066 (cytochrome b gene sequencing).
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Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, Louisiana 70433
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Research East 113, 41 Avenue Louis Pasteur, Boston, Massachusetts 02115
ABSTRACT
The simian immunodeficiency viruses (SIV) naturally infect a wide range of African primates, including African green monkeys (AGM). Despite moderate to high levels of plasma viremia in naturally infected AGM, infection is not associated with immunodeficiency. We recently reported that SIVagmVer90 isolated from a naturally infected vervet AGM induced AIDS following experimental inoculation of pigtailed macaques. The goal of the present study was to evaluate the replication of this isolate in two species of AGM, sabaeus monkeys (Chlorocebus sabaeus) and vervets (C. pygerythrus). Inoculation of sabaeus AGM with SIVagmVer90 resulted in low and variable primary and set-point viremia (<102 to 104 copies/ml). In contrast, inoculation of vervet AGM with either SIVagmVer90 or blood from a naturally infected vervet (Ver1) resulted in high primary viremia and moderate plateau levels, similar to the range seen in naturally infected vervets from this cohort. CD4+ T cells remained stable throughout infection, even in AGM with persistent high viremia. Despite the lack of measurable lymphadenopathy, infection was associated with an increased number of Ki-67+ T cells in lymph node biopsies, consistent with an early antiviral immune response. The preferential replication of SIVagmVer in vervet versus sabaeus AGM shows that it is critical to match AGM species and SIV strains for experimental models of natural SIV infection.
INTRODUCTION
The simian immunodeficiency viruses (SIV) are a diverse group of lentiviruses that infect a wide variety of African nonhuman primates, including sooty mangabey monkeys (SM) and African green monkeys (AGM). Molecular studies of SIV strains have demonstrated phylogenetic lineages that are specific for the primates of origin. For example, SIV lineages have been identified that are specific for SM (SIVsm) and AGM (SIVagm). The initial identification of distinct subtypes of SIVagm (SIVagmGri, -Ver, -Sab, and -Tan) specific to the four geographically separated AGM species, namely, grivet (7), vervet (1, 22, 45), sabaeus (21), and tantalus (18, 29) monkeys (Chlorocebus aethiops, C. pygerythrus, C. sabaeus, and C. tantalus, respectively), suggested that these viruses have coevolved with their primate host species over a long period of time (3, 29). Despite high seroprevalence rates in feral and captive populations, natural infections of African primates with their own SIV strains are not generally associated with immunodeficiency (16). While it is difficult to assess the effect of SIV infection in wild populations, large numbers of AGM and SM have been studied in captivity over long periods of time. The conclusion of these various studies is that SIV infections of natural host species are generally asymptomatic. CD4+ T-cell depletion has been observed in a few isolated cases, including a naturally infected mandrill (36), an African green monkey (44), and, most recently, a sooty mangabey (27), suggesting that in some circumstances these viruses may produce AIDS after long incubation periods. These studies suggest that African primates are not immune to the pathogenic effects of primate lentiviruses under specific circumstances. SIV strains are not inherently apathogenic, since experimental cross-species transmission to Asian macaque species results in an AIDS-like syndrome (17, 46). The mechanistic basis for the lack of disease in naturally infected species such as AGM may therefore provide insight into the pathogenic mechanism of AIDS and suggest novel therapeutic strategies.
An intriguing feature of natural infections of sooty mangabeys (SIVsm), AGM (SIVagm), and mandrills (SIVmnd) is the failure to develop AIDS in the face of persistent moderate to high viral loads (4, 11, 35, 37, 41). Experimental infections of mandrills with SIVmnd (33, 35) and of sabaeus AGM with SIVagm/Sab (6) resulted in high levels of primary viremia followed by partial containment of viremia. Therefore, the lack of disease in these SIV-infected African monkeys is clearly not due to suppression of viremia such as that observed in long-term nonprogressors among HIV-infected humans (5). Despite vigorous studies of both sooty mangabeys and African green monkeys, the mechanisms whereby these animals avoid AIDS are not clear (4, 13, 28, 39, 41). Therefore, experimental animal models that recapitulate the virologic and immunologic features of infection are essential. The goal of the present study was to establish an animal model using our previously characterized SIVagmVer90 isolate by investigating the viral replication of SIVagmVer90 in two species of AGM, vervets and the more readily available Caribbean sabaeus AGM.
MATERIALS AND METHODS
Animals and viruses. The naturally infected African green monkey (AGM90) that was the source of SIVagmVer90 was imported from Kenya in 1987 and phenotypically appeared to be a vervet monkey (11). The virus was isolated from the mesenteric lymph nodes of monkey AGM90 by coculture of viably frozen mononuclear cells with macaque peripheral blood mononuclear cells (PBMC). Viruses were titrated for infectivity in the susceptible cell line CEMss. Six sabaeus AGM and four vervet AGM were inoculated intravenously with 1,000 50% tissue culture infective doses (TCID50) of SIVagmVer90. Animals were maintained in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals under an NIAID-approved animal study protocol (32) and were housed in a biosafety level 2 facility using biosafety level 3 practices. The vervet AGM utilized for this study were imported from Tanzania and screened for SIV infection by Western blotting, radioimmunoprecipitation, virus isolation from PBMC, and measurement of plasma viral RNA (vRNA) loads. One seropositive vervet AGM (Ver1) was used as a source of SIVagm-infected blood for transfusion of 2 ml of heparinized blood into four seronegative vervets. A limiting 10-fold-dilution coculture of PBMC isolated from AG1 blood collected at the time of transfusion was performed with CEMss cells. Virus isolation required 106 PBMC, and thus this inoculum represented approximately 50 TCID.
Genetic characterization of vervet AGM by cytochrome b gene sequencing. In order to confirm the species of vervet AGM, DNAs were extracted from 300 μl of whole blood from four SIV-seronegative monkeys from the Tanzanian cohort (Ver7, Ver9, Ver13, and Ver23). A QIAamp blood kit (QIAGEN, Valencia, CA) was used to extract the DNAs, which were eluted into 60 μl of H2O and stored at –20°C. A 267-bp region of the cytochrome b gene, representing bases 14841 to 15149 of the human mitochondrial genome (2), was amplified using primers L14841 and H15149 (20). One microgram of DNA was used in a 100-μl PCR mix for PCR in a Perkin-Elmer PE 9700 thermocycler. The PCR conditions were as follows: 2 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C, with a final extension of 5 min at 72°C. Following purification with a QIAquick PCR purification kit (QIAGEN), the PCR products were cycle sequenced and analyzed on an ABI 377 automated DNA sequencer. Cytochrome b gene sequences from different species of AGM were aligned with that from a grivet (GenBank accession no. AY863426) and with sequences from other African nonhuman primates retrieved from GenBank. Sequences were aligned using ClustalW 1.8. Unrooted maximum parsimony trees were inferred using PAUP, version 4.0b8, and were uniformly weighted and unordered.
Virus isolation. Viruses were isolated from PBMC of experimentally infected AGM. Ficoll-separated PBMC were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, stimulated with 5 μg per ml phytohemagglutinin, and cultivated for 3 days in the presence of 20 units per ml of interleukin-2. Subsequently, 5 x 106 PBMC were cocultivated with CEMss cells for 6 weeks, with biweekly feeding, and the culture supernatant was monitored for reverse transcriptase (RT) activity as previously described (10).
Plasma vRNA assay. A real-time RT-PCR assay for quantitation of viral RNA in plasma was performed as previously described (11), using methodology based on the 7700 sequence detection system (Applied Biosystems, Foster City, CA) that was used previously for SIVsm/mac-specific real-time PCR (43). Briefly, forward and reverse primers to amplify a 122-bp fragment and an internal fluorogenic probe were generated based on the SIVagm155 sequence (GenBank accession no. M29975), as follows: AgmF, 5'-GTC CAG TCT CAG CAT TTA CTT G-3' (7981); AgmR, 5'-CGG GCA TTG AGG TTT TTC AC-3' (nucleotide 8090); and probe, 5'-R-CAG ATG TTG AAG CTG ACC ATT TGG GQ-3' (nucleotide 8041), where R indicates a 6-carboxyfluorescein group and Q indicates a 6-carboxytetramethylrhodamine group conjugated through a linker arm nucleotide linkage. Previous studies have shown that this primer/probe set amplifies divergent SIVagmVer isolates (11). Sequence analysis of env clones from Ver1 showed that sequences in the areas of both primers were conserved. There is one mismatch of Ver1 env with the probe sequence which is shared with SIVagmVer90. The high-performance liquid chromatography-purified probe was obtained from Applied Biosystems/Perkin-Elmer (Foster City, CA). Plasma samples for analysis were collected by using EDTA as an anticoagulant and were stored in a –80°C freezer until analysis. Plasma viral RNAs were isolated using a QIAmp viral RNA kit (QIAGEN, Valencia, CA). Sense RNA transcribed using T7 polymerase from an EcoRI-linearized plasmid created from a 1.9-kb fragment (HindIII/HincII) of the envelope of pSIVagm9063-2 cloned into the pTRI-10 [poly(A)] plasmid vector was used as a standard for the assay. RT reactions were performed in 96-well plates, and the mixtures contained identical concentrations of the following components in RNase-free water: random hexamers (2.5 μM; Promega, Madison, WI), 5.0 mM MgCl2, a 1.0 mM concentration of each deoxynucleoside triphosphate, 5.0 mM dithiothreitol, and 20 U Superscript RT.
SIV-specific antibodies. Serology for antibodies to SIVagm was performed by Western blot analysis, as previously described (17). Briefly, the virus was pelleted from the cell-free supernatant of SIVagmVer90-infected CEMss cells, and virus lysates were electrophoresed in a polyacrylamide gel, transferred to nitrocellulose, and used as an antigen to react with macaque plasma. SIV antibodies were detected by using an anti-human immunoglobulin G antibody conjugated with alkaline phosphatase (Amersham, Piscataway, N.J.). Bands were detected upon the addition of the BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) phosphatase substrate system (KPL Laboratories, Gaithersburg, MD). The serologic status of the wild-caught vervets was confirmed by radioimmunoprecipitation as previously described (14), using CEMss cells infected for a short term with SIVagmVer90 as the source for cell lysates.
Absolute CD4+ T-cell counts. CD4+ T cells were analyzed sequentially throughout infection by flow cytometry. Briefly, EDTA-anticoagulated 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 a fluorochrome. The monoclonal antibodies used to identify CD4+ T-cell subsets were L200-allophycocyanin (BD Biosciences, San Jose, CA) for vervets and OKT4a-fluorescein isothiocyanate (Ortho Diagnostic Systems, Raritan, N.J.) for sabaeus monkeys. Following staining, erythrocytes were lysed, and leukocytes were fixed in 1% paraformaldehyde and analyzed using either a Becton Dickinson FACSVantage SE DiVa cell sorter, for specimens from vervets, or a Coulter Epics 753 cell sorter, for specimens from sabaeus monkeys. To determine the absolute CD4+ T-cell counts, the percentages of CD4+ T lymphocytes were multiplied by the absolute lymphocyte counts obtained using the ADVIA 120 hematology system (Bayer, Tarrytown, NY).
SIV-specific in situ hybridization. Nonradioactive in situ hybridization (ISH) for SIV expression was performed on formalin-fixed, paraffin-embedded lymph nodes, utilizing sense or antisense digoxigenin-labeled riboprobes that spanned the entire SIVagm9063-2 genome, as previously described (17). Briefly, sections were deparaffinized and rehydrated with water, pretreated with 0.2 N HCl and proteinase K, and then hybridized overnight at 51°C with either the antisense or the sense riboprobe. The riboprobe consisted of a mixture of probes conjugated with digoxigenin-UTP (Lofstrand Labs, Ltd., Gaithersburg, Md.). The hybridized sections were washed in standard posthybridization buffers and RNase A solutions (RNase A [Sigma] and RNase T1 [Roche Molecular Biochemicals, Indianapolis, Ind.]). The sections were blocked with 3% normal sheep and horse sera 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, incubated with NBT/BCIP (Vector Laboratories, Ltd., Burlingame, Calif.) for 10 h, and visualized with a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, N.Y.).
Immunohistochemistry. Ki-67 expression was assessed in sequential formalin-fixed, paraffin-embedded lymph node samples by immunohistochemical staining as previously described (19). Samples were incubated with Ki-67 (DAKO) and goat anti-rabbit immunoglobulin G-Alexa fluor 488 (Invitrogen). The stained sections were rinsed, counterstained with hematoxylin, covered, and photographed with a Zeiss Axiophot microscope equipped with a Cool Snap digital camera (Biovision). Sections were analyzed by segmenting (counting) the total number of SIV-positive cells by ISH (IP Lab software) from three representative fields of view of the paracortical region of each section. The total number of Ki-67+ cells per region of interest was calculated and imported into Prism 4, and the means and standard errors of the means (SEM) were graphed.
Sequence analysis. PCR was used to amplify a 370-bp fragment of the transmembrane glycoprotein-encoding region of envelope from 500 ng of genomic DNA extracted from CEMss cell isolates of SIVagm from 12 SIV-seropositive AGM from the Tanzanian cohort. Primers for amplification were designed based on an alignment of published SIVagm/Ver isolates and had the following sequences: forward primer, 5'-CCT CAA TGC CCG CGT CAC AGC-3'; and reverse primer, 5'-CTC CTG CGT TGT CTG GCT GTC-3'. The resulting products were purified by electrophoresis in a 2% agarose gel, extracted from the gel using a QIAquick PCR purification kit (QIAGEN), and sequenced directly using the PCR primers and a Terminator cycle sequencing kit for fluorescence-based sequencing (Applied Biosystems, Warrington, United Kingdom). Phylogenetic analysis was performed using neighbor joining calculated from a Kimura two-parameter distance matrix (MacVector).
Statistical methods. The differences in viral loads and CD4+ T-cell numbers between different groups of monkeys at various time points were determined by the Mann-Whitney test using GraphPad Prism, version 4, software (San Diego, CA).
RESULTS
Selection and characterization of study animals. Six Caribbean green monkeys were imported from St. Kitts and screened serologically for SIV and simian T-cell leukemia virus type 1 (STLV-1) infection. As expected, all of these animals were seronegative for SIVagm. These animals evolved from a founder population imported from West Africa over 300 years ago and are presumed to be of the sabaeus species based upon their origin and phenotypic appearance. As shown in Fig. 1, sequence analysis of cytochrome b genes from Caribbean AGM are also consistent with their classification as sabaeus AGM (I. Pandrea, C. Apetrei, J. Dufour, N. Dillon, J. Barbercheck, M. Maetzger, B. Jacquelin, R. Bohm, P. A. Marx, F. Barre-Sinoussi, V. Hirsch, M. C. Muller-Trutwin, A. A. Lackner, and R. S. Veazey, submitted for publication). Cytochrome b gene analysis was not performed on the sabaeus monkeys used in the present study since they were animals obtained from the same source as the Caribbean sabaeus AGM used for Fig. 1.
Twenty-four wild-caught AGM were imported from Tanzania, a part of the known geographical habitat of vervets. Phylogenetic analysis of cytochrome b genes from four representative animals from this cohort confirmed that they were genetically related to other AGM but formed a phylogenetic cluster distinct from sabaeus, tantalus, and grivet monkeys (Fig. 1), consistent with their classification as vervets. Western blot analysis of plasma samples identified 12 of the vervets as SIV seropositive, and viral RNA was detected in 11 of these by a vervet-specific real-time RT-PCR assay. Viral RNA was not detected in the plasma of one of the vervets (Ver15), presumably due to a mismatch in the primer/probe sequences. Plasma vRNA levels were consistent in individual animals upon six repeated monthly assays, with mean values for the individuals ranging from 3,400 to 217,000 copies/ml of plasma (Table 1). A 370-nucleotide fragment of the transmembrane portion of env was amplified by PCR from DNAs extracted from viruses isolated from PBMC of the seropositive vervets. This region spans the extracellular variable region of the transmembrane protein and could be readily amplified using highly conserved primers. As shown in Fig. 2, each of the envelope sequences was distinct, but the sequences clustered phylogenetically with previously characterized SIVagm isolates from vervet monkeys (SIVagmVer90 and SIVagmVer155) and were distinct from representative SIVagm isolates from the other three species of AGM, i.e., grivet, sabaeus, and tantalus monkeys. Eight of the 12 seronegative vervets were chosen for subsequent experimental inoculation with SIVagmVer, and one vervet (Ver1) with high viremia was chosen as a source of blood for the inoculum.
Inoculation of sabaeus and vervet AGM with SIVagmVer. Four vervets (Ver20, Ver21, Ver22, and Ver24) and six sabaeus monkeys (Sab951, Sab954, Sab955, Sab964, Sab005, and Sab038) were inoculated with the same dose and stock of cryopreserved SIVagmVer90 used in our previous study of pig-tailed (PT) macaques (12). An additional four vervets (Ver2, Ver8, Ver14, and Ver19) were inoculated with 1 ml of EDTA-anticoagulated blood from the naturally infected vervet, Ver1. All of the animals were inoculated intravenously and monitored sequentially by virus isolation from PBMC and lymph node biopsies, flow cytometric analysis of peripheral blood CD4+ T lymphocytes, plasma viral RNA levels, SIV-specific ISH of lymph nodes, and SIV serology by Western blot assay.
All animals became infected, as evidenced by the isolation of infectious virus from PBMC or lymph nodes by cocultivation with CEMss cells following stimulation with phytohemagglutinin and interleukin-2. Virus isolation from vervet PBMC was highly reproducible at sequential time points throughout infection. In contrast, virus isolation from sabaeus monkeys inoculated with SIVagmVer90 was inconsistent (Table 2). Virus was only occasionally isolated from PBMC samples of two of the sabaeus AGM (Sab951 and Sab954). For example, virus was only isolated from lymph node biopsies collected from Sab951 at 1 and 4 weeks postinoculation and not from PBMC at any other time points. These data suggested more robust replication of SIVagmVer viruses in vervet monkeys than in sabaeus monkeys.
High viremia in vervets but not sabaeus monkeys inoculated with SIVagmVer. For a more quantitative assessment of virus replication, plasma viral RNA was measured using a real time RT-PCR assay developed specifically for vervet isolates of SIVagm (11). As shown in Fig. 3A and B, primary viremia was consistently high in vervets inoculated with either SIVagmVer90 or SIVagmVer1 (106 to 108 copies/ml of plasma). Peak viremia was slightly higher for vervets that received blood from Ver1, and the peak was slightly delayed compared to that for vervets inoculated with SIVagmVer90 (10 days versus 7 days). This delay in peak viremia in vervet AGM inoculated with AG1 blood may have been due to the difference in the sizes of the inocula (50 versus 1,000 TCID). Primary infection of vervets was followed by an abrupt decline in plasma viremia by 4 weeks postinoculation in all animals and then the establishment of a stable plateau level of 104 to 106 copies/ml. Therefore, the replication kinetics of SIVagmVer90 and SIVagmVer1 were similar in vervet monkeys.
A much wider range in primary viremia was observed for the sabaeus monkeys (Fig. 3C and D), ranging from undetectable (Sab951) to as high as 106/ml (Sab964). The peak of primary viremia was also delayed, to 2 to 3 weeks postinoculation, compared to 1 week postinoculation for the vervets. The two sabaeus monkeys (Sab955 and Sab964) with the most consistent isolation of SIV from PBMC (Table 2) also had the highest levels of primary viremia. An abrupt decline in plasma viremia was observed in all sabaeus monkeys by 8 to 12 weeks postinoculation, and all of the animals except for Sab951 maintained detectable levels of plasma vRNA throughout the 100-week period of observation. As observed during primary infection, the range in chronic viremia was very broad and dependent on the individual, ranging from undetectable (<102) to 106 copies/ml of plasma.
A comparison of sequential mean viral RNA levels (Fig. 3D) among AGM inoculated with SIVagmVer clearly demonstrated that SIVagmVer90 replication was less robust in sabaeus monkeys than in vervets, suggesting a specific host adaptation. Primary viremia was significantly lower (P < 0.01) in sabaeus monkeys than in vervets infected with SIVagmVer90, with no overlap between values. Set-point viremia was also significantly lower in the sabaeus monkeys (P < 0.01), ranging from <100 to 5,700 copies/ml, than in the vervet AGM (8,000 to 300,000 copies/ml). In contrast, the viral replication kinetics of SIVagmVer90 and SIVagmVer1 in vervets were similar. The peak of primary viremia in vervets inoculated with SIVagmVer1 was slightly delayed relative to that in vervets inoculated with SIVagmVer90 (Fig. 3D). However, there was no significant difference in viral loads in the postacute period of infection in vervets inoculated with either SIVagmVer90 or Ver1 blood (P = 0.32).
Stable blood CD4+ T-lymphocyte numbers in SIVagm-infected AGM. CD4+ T lymphocytes in the peripheral blood of AGM were monitored over a period of 90 weeks following inoculation (Fig. 4). A transient decline in CD4+ T cells was observed during primary infection in a subset of the animals. CD4+ T cells in peripheral blood remained stable throughout the entire period of observation, with no differences observed between the different species inoculated. All of the AGM remained asymptomatic, although one vervet (Ver21) was euthanized at 70 weeks postinoculation due to non-AIDS related causes (self-mutilation). The lack of peripheral CD4+ T-cell depletion in both vervets and sabaeus monkeys in the present study contrasted with the marked depletion we observed previously in PT macaques inoculated with SIVagmVer90 (12).
Robust envelope-specific antibody responses in AGM. SIV-specific antibody responses of vervet and sabaeus AGM inoculated with SIVagmVer90 were monitored by Western blotting using SIVagmVer90 as the viral antigen. As shown in Fig. 5, all but one of the AGM (Sab951) developed SIV-specific antibody responses. This particular animal exhibited the most inconsistent virus isolation from PBMC and lymph nodes, and viral RNA was only detected at one time point, all of which are consistent with a transient or subclinical infection. All other animals responded with robust levels of envelope-specific antibody, but with much weaker responses to Gag or other viral antigens. The predominance of envelope-specific antibody responses has been observed previously by us and others in naturally infected AGM (11, 31), and its significance is not clear in terms of its relationship to the lack of disease in AGM.
SIV-specific ISH and immunohistochemical analysis of lymph nodes. SIV-specific ISH was performed on sequential lymph node biopsies sampled at 1, 2, and 4 weeks postinoculation (Fig. 6). A comparison of SIV expression in lymph nodes in vervets and sabaeus monkeys inoculated with SIVagmVer90 revealed robust infection in lymphoid tissues of the vervets. A similar level of expression was observed in the vervets inoculated with SIVagmVer1 (data not shown). Representative fields are shown in Fig. 6B to demonstrate the large number of SIV-expressing cells in lymph node biopsies collected at 1 week from vervets inoculated with SIVagmVer90 and the subsequent decrease by 2 weeks. This high level of expression contrasted with the low expression of SIV in the lymph nodes of sabaeus monkeys inoculated with SIVagmVer90. SIV-expressing cells were only observed in one lymph node biopsy collected from H964 at 1 week postinoculation (peak viremia). Thus, the expression of SIV in lymph nodes reflected the differences in primary viremia observed between vervets and sabaeus monkeys inoculated with a common SIVagmVer90 strain.
The lymph nodes of both vervets and sabaeus AGM remained constant in size throughout infection, without evidence of paracortical expansion. This contrasts with the marked lymphadenopathy observed in SIVagmVer-inoculated PT macaques (12, 16). We focused on analyzing the vervets since the reduced viral replication observed in the sabaeus monkeys suggested that they might not be reflective of natural infection. To assess the extent of immune activation occurring with lymph nodes during the first month of infection, Ki-67 expression was assessed in formalin-fixed lymph nodes collected preinoculation and 1, 2, and 4 weeks after infection from the SIVagmVer90-inoculated vervets. As shown graphically in Fig. 7A, the number of Ki-67+ cells in the paracortical areas of the lymph nodes increased significantly following infection (P < 0.01), reaching peak levels by 2 weeks and subsiding by 4 weeks. As evident in a representative section in Fig. 7B, preinoculation lymph nodes were relatively quiescent, with small numbers of Ki-67+ cells in the paracortical regions (P) and rare germinal centers (GC). By 2 weeks postinoculation, developing germinal centers stained intensely for Ki-67, consistent with the expansion of B lymphocytes (Fig. 7B, bottom panel). Confocal analysis showed that Ki-67+ cells in the paracortex (data not shown) represent a mixture of CD4+ and CD8+ T cells, with a predominance of CD8+ T cells.
DISCUSSION
This study extends our previous evaluation of a primary SIVagmVer isolate in PT macaques, a pathogenic model (12) of the natural AGM host for this virus. The levels of chronic viremia established in vervet AGM in the present study were similar to the levels observed in the naturally infected cohort of vervets and to previous reports of natural SIVagm infection in AGM (4, 11). In addition, the pattern of primary and chronic viremia observed in vervet monkeys infected with SIVagmVer90 was similar to that in previous reports of sabaeus monkeys experimentally inoculated with SIVagmSab (34). Virus replication levels following infection with SIVagmVer90 and SIVagmVer1 (blood inoculum) were similar in vervets, suggesting that SIVagmVer90 has not been significantly modified in biologic terms by passage in macaque PBMC. However, the magnitude of viremia of SIVagmVer90 was dependent on the particular species of AGM used for experimental infection. Despite the fact that the sabaeus and vervet species exhibit only minor differences in genes that are critical for SIV infection, such as the CD4 and CCR5 genes (8, 30), SIVagmVer replicated significantly more efficiently in vervets than in sabaeus monkeys. Although viral replication of SIVagmVer in vervets and sabaeus monkeys differed, the clinical outcomes were similar. All animals remained asymptomatic for 90 weeks following infection. Despite different levels of persistent viremia among the AGM, peripheral blood CD4+ T-cell levels were remarkably stable.
The present study confirms recent observations of significant differences in the replication of specific SIVagm isolates in different species of AGM. A previous study showed that SIVagmSab replicated efficiently in both sabaeus and vervet AGM (34) and that SIVagmVer replicated inefficiently in both sabaeus and vervet AGM, suggesting virus-specific replication. This contrasts with the results of our study, where we observed robust replication of SIVagmVer90 and SIVagmVer1 in vervet AGM but inefficient replication of these viruses in sabaeus AGM; thus, virus replication was dependent on the host species inoculated with SIVagmVer. Efficient replication was only observed in the homologous host. Although the previous study observed virus-specific replication differences, it is clear from these two studies that the virus and host species must be carefully considered and matched to the virus for natural history and virologic studies of SIVagm in vivo. We can only speculate on the host factors responsible for the host restriction of SIVagmVer in sabaeus monkeys. Since the restriction in viral replication occurs during primary infection, the mechanisms are likely to be related to innate immunity or host restriction factors, such as APOBEC3G (9, 15) or TRIM5alpha (23, 38, 42). Alternatively, factors involved in virus entry, such as CCR5 and CD4, may be responsible; this hypothesis seems unlikely since there are only minor differences in the sequences of CD4 and CCR5 (8, 25, 26, 30) between these two species.
Finally, the kinetics of early viremia in vervet monkeys inoculated with SIVagmVer was indistinguishable from that of viremia observed in PT macaques inoculated with SIVagmVer90 (12). This study confirms recent studies of SIVsm infections of sooty mangabeys and macaques that showed robust replication kinetics for both asymptomatic and pathogenic infections (40). Since viral replication was similarly robust in pathogenic and natural asymptomatic infections, it is likely that significant differences in host responses to SIV infection are responsible for divergent disease outcomes. Previous studies have suggested that the levels of virus-induced immune activation may be significantly lower in naturally infected host species than in hosts which develop AIDS (40, 41). In the present study, we demonstrated significant proliferation (as assessed by Ki-67 expression in lymph nodes) in SIVagmVer-inoculated vervet AGM. Such an increase in Ki-67+ cells is probably the result of a "normal" immune response that would be expected in any viral infection. This establishes that these animals are not inherently unresponsive to SIV infection and suggests that qualitative and dynamic differences in their reactions to infection compared to those in pathogenic macaque models may be responsible, in part, for their lack of disease. A side-by-side comparison with SIVagmVer-infected PT macaques will be necessary to evaluate whether this parameter differs in the two species. A preliminary analysis of immune activation in SIVagmVer-inoculated PT macaques showed similar kinetics of Ki-67-expressing cells in lymph nodes (data not shown). However, the numbers of Ki-67+ cells were always higher in PT macaques, even prior to SIV inoculation (data not shown). This finding is in agreement with comparative studies of sooty mangabeys and macaques infected with SIVsm, where greater numbers of Ki-67+ cells were observed in T-cell areas of macaque lymph nodes at 4 weeks postinfection (40).
Recent studies of AGM experimentally inoculated with SIVagm may shed some light on the potential mechanism for the lack of hyperimmune activation observed in AGM. Infection of sabaeus AGM with SIVagmSab was found to be associated with an early anti-inflammatory profile of gene expression, characterized by the induction of transforming growth factor 1 and FoxP3 and an increase in regulatory T cells (24); this profile differs from those observed for SIVmac infection of macaques and human AIDS, which are both associated with aberrant chronic T-cell activation. These data suggest that this early environment promotes the generation of regulatory T cells, which could dampen or prevent chronic hyperactivation. A direct comparison of AGM and macaques inoculated with a common virus will therefore be highly informative in determining the significance of immune activation in CD4+ T-cell-induced cell death and AIDS. Other potential mechanisms that will also be investigated include differences in cytopathic effects, CD4 regenerative ability, and disparity in target cell type or availability between the two hosts.
In summary, different species inoculated with a common virus (SIVagmVer) had the following distinct outcomes: (i) high viremia with disease in PT macaques, (ii) high viremia with no disease in vervet AGM, and (iii) moderate to low viremia with no disease in sabaeus AGM. SIVagmVer appears to be specifically adapted to vervet monkeys, in contrast to sabaeus AGM, with these species differing in terms of permissiveness for infection. In contrast, PT macaques and vervet AGM appear to be relatively equivalent in terms of their permissiveness for infection. However, these two species differ widely in terms of disease induction, with macaques developing AIDS (12) and vervets remaining healthy.
Preliminary studies suggest that vervets and macaques differ with respect to the extent of immune activation associated with infection, similar to observations for SIVsm infections of sooty mangabeys and macaques (40). Together, these data suggest that the level of immune activation may be a key determinant of disease progression. Experimental infections of vervet AGM and PT macaques with SIVagmVer90 will provide a valuable model system to explore differences in immunologic responses and target cells that differentiate asymptomatic natural infections from AIDS.
ACKNOWLEDGMENTS
We thank S. Whitted and R. Goeken of LMM, NIAID, and Cristian Apetrei of Tulane National Primate Research Center for technical assistance and Russell Byrum of Bioqual Inc. for conducting the animal studies.
This study was supported by the intramural program of the National Institute of Allergy and Infectious Diseases, NIH, and by NIH grants RO1 AI065335 (flow cytometric studies) and RO1 AI064066 (cytochrome b gene sequencing).
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