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High levels of SIVmnd-1 replication in chronically infected Mandrillus sphinx
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Virology 317 (2003) 119 –127
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High levels of SIVmnd-1 replication in chronically infected Mandrillus sphinx Ivona Pandrea,a,b,1 Richard Onanga,a,1 Christopher Kornfeld,c Pierre Rouquet,a Olivier Bourry,a Stephen Clifford,a Paul T. Telfer,a Kate Abernethy,a Lee TW White,a Paul Ngari,a Michaela Mu¨ller-Trutwin,c Pierre Roques,a,d Preston A. Marx,b Francois Simon,a,e and Cristian Apetreia,b,* a
Laboratoire de Virologie, UGENET, SEGC, Re´serve de la Lope´, Centre de Primatologie, Centre International de Recherches Me´dicales, BP769, Franceville, Gabon b Tulane National Primate Research Center, Covington, LA 70433, USA c Unite´ de Biologie des Re´trovirus, Institut Pasteur, 29, Rue Dr. Roux, 75724 Paris Cedex 15, France d Service de Neurovirologie, Commisariod a` l’Energze Atomygue, 92265 Foulenay aux Roses, France e Hopital Charles Nicolle, 76031 Rouen, France Received 14 May 2003; returned to author for revision 17 June 2003; accepted 14 August 2003
Abstract Viral loads were investigated in SIVmnd-1 chronically infected mandrills and the results were compared with those previously observed in other nonpathogenic natural SIV infections. Four naturally and 11 experimentally SIVmnd-1-infected mandrills from a semi-free-ranging colony were studied during the chronic phase of infection. Four SIVmnd-1-infected wild mandrills were also included for comparison. Twelve uninfected mandrills were used as controls. Viral loads in all chronically infected mandrills ranged from 105 to 9 ⫻ 105 copies/ml and antibody titers ranged from 200 to 14,400 and 200 to 12,800 for anti-V3 and anti-gp36, respectively. There were no differences between groups of wild and captive mandrills. Both parameters were stable during the follow-up, and no clinical signs of immune suppression were observed. Chronic SIVmnd-1-infected mandrills presented slight increases in CD20⫹ and CD28⫹/CD8⫹ cell counts, and a slight decrease in CD4⫹/CD3⫹ cell counts. A slight CD4⫹/CD3⫹ cell depletion was also observed in old uninfected controls. Similar to other nonpathogenic models of lentiviral infection, these results show a persistent high level of SIVmnd-1 replication during chronic infection of mandrills, with minimal effects on T cell subpopulations. © 2003 Elsevier Inc. All rights reserved. Keywords: SIVmnd-1; Pathogenesis; Mandrills; Viral load; Lymphocyte subsets
Introduction More than 30 simian immunodeficiency viruses (SIVs) naturally infect nonhuman African primates (Apetrei et al., 2004). Their overall seroprevalence in the wild is very high (estimated 18 –30%) (Jolly et al., 1996; Chen et al., 1996; Souquiere et al., 2001; Peeters et al., 2002). Although they were named SIV because of their structural and biological
* Corresponding author. Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433. Fax: ⫹1-985-871-6248. E-mail address: [email protected] (C. Apetrei). 1 These authors contributed equally to this study. 0042-6822/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2003.08.015
resemblance to the human immunodeficiency virus (HIV), SIV infections are mostly nonpathogenic in their natural hosts (Beer et al., 1998; Rey-Cuille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Silvestri et al., 2003). A few exceptions have been documented in mandrills and African green monkeys (Pandrea et al., 2001; Souquiere et al., 2001; Traina-Dorge et al., 1992). Disease is probably a rare consequence because of long-term adaptation of SIV to its natural African primate hosts, which is reflected in continuous and sustained viral replication with minimal effects on immune function (Kaur et al., 1998; Silvestri et al., 2003). There is evidence showing that SIVs have pathogenic
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potential. Cross-species transmission of SIVsm to macaques results in increased pathogenicity and induction of simian acquired immunedeficiency syndrome (sAIDS) (Letvin et al., 1985; Murphey-Corb et al., 1986). HIV-1 and HIV-2, which have simian origins, are responsible for the AIDS pandemic (Peeters et al., 1989; Gao et al., 1999; Chen et al., 1996). Experimental transmission of SIVagm from African green monkeys (AGMs) and SIVIhoest from I’hoesti monkeys to pig-tailed macaques has also shown pathogenic potential and immunosuppression (Hirsch et al., 1995, 1999). In these cases, high viral loads induce massive activation and destruction of the immune system (Alimonti et al., 2003). Therefore, plasma viral load is considered the best predictor of the clinical outcome in humans and macaques (Mellors et al., 1996; Lifson et al., 1997). In this study, we have investigated the relationship between SIV and its natural host in a new model, SIVmnd-1 chronic infection of mandrills. The probable origin of SIVmnd-1 was reported to be from Cercopithecus lhoesti solatus to mandrills (Hirsch et al., 1999; Beer et al., 1999). This observation raised the question as to whether or not mandrills should be considered as long-term natural hosts of SIVmnd-1 and whether the course of infection would be the same as in other nonpathogenic infections. The preponderance of evidence indicates that mandrills are a natural host because recent studies have shown that SIVmnd-1 is highly prevalent in wild mandrills, suggesting that cross-species transmission was an ancient event and that the virus infects this species naturally (Souquiere et al., 2001). SIVmnd-1 replicates to high levels during experimental primary infection in mandrills and this replication has only slight and transitory effects on the dynamics of immune cells (Onanga et al., 2002). This pattern is very similar to those observed during the primary infection of other nonhuman primate species (Diop et al., 2000; Kaur et al., 1998). Here we address the hypothesis as to whether or not mandrills represent an intermediate level between pathogenic and nonpathogenic SIV infections or whether or not the virus is adapted to the mandrill host. Only one SIVmnd-1 strain (GB-1) is available and it was adapted in vitro and reported to use CXCR4 as the main coreceptor, which is highly unusual for SIV (Schols and De Clerq, 1998; Beer et al., 2001). Therefore, SIVmnd-1 (GB-1) may have a higher pathogenic potential compared to SIVs which use CCR-5 as the major coreceptor (Marx and Chen, 1998). However, SIVmnd-1 GB1 recovered during the primary infection used CCR5 and Bonzo and not CXCR4 (R. Onanga, unpublished observation). As chronic infection reflects the long-term natural history of SIV, the aim of the present study was to measure the dynamics of RNA plasma viral load, anti-SIVmnd-1 antibody titers, and lymphocyte subsets in mandrills chronically infected with SIVmnd-1 to compare these data with those previously observed in other nonpathogenic models. Data from wild mandrills are also presented for comparison.
Results Clinical and immunological follow-up of chronically infected SIVmnd-1 mandrills No clinical signs of AIDS were observed during annual follow-ups of chronically, naturally infected mandrills (17B, 17D, 17G, 17D2) in captivity. Eleven experimentally infected animals (n ⫽ 11) were examined every 3 months. No follow-up was possible for the wild mandrills, but all were clinically normal at the time of sampling. Viral load quantification in experimentally infected mandrills In HIV-infected humans and SIV-infected rhesus macaques the quantification of plasma viral RNA provides a measure of the magnitude of virus replication (Mellors et al., 1997; Lifson et al., 1997). For viral RNA loads in plasma, a QC-RT-PCR assay specific for SIVmnd-1 was developed (Onanga et al., 2002) using primers MP5/MP6 from a conserved region in integrase of SIVmnd-1 GB-1 (Nerrienet et al., 1998). There were no mismatches (Onanga et al., 2002). Our previous studies on the acute phase of SIVmnd-1 infection showed that high viral loads obtained by QC-RT-PCR directly correlated with both p24 Ag titers and plasma RT activity (Onanga et al., 2002). Experimentally infected mandrills included in this study were mandrills from a primary infection experiment (12A4, 2C2, 10G, 12C2) inoculated intravenously (iv) with 3000 TCID50, and dams in a breastfeeding transmission experiment (5I, 5J, 12A3, 12A7, 6B, 16C), which received 300 TCID50 iv of the same stock. Mandrill 16E received 10 ml of blood from the mandrill naturally infected with GB-1 (F17). This virus was titered at 4 TCID50 on SupT1 cells, a value which corresponded to 896,000 RNA copies/ml (Onanga et al., 2002). In experimentally infected mandrills, the viral set point was reached between days 30 and 60 postchallenge (Onanga et al., 2002). In this present study we quantified the SIVmnd-1 RNA loads in 19 samples collected between days 180 and 540 postchallenge from all 11 experimentally infected mandrills (Table 1). SIVmnd-1 plasma viral loads are presented in Table 1. Although results obtained at days 180 and 360 for mandrills 12A4, 16E, 2C2, and 10G have been presented elsewhere, these points are also showed in Table 1 for comparison. Viral RNA levels ranged from 18,000 to 334,000 copies/ml, similar to the range observed in SIVsm-infected sooty mangabeys (ReyCouille et al., 1998; Ling et al., 2003; Silvestri et al., 2003) and AGMs (Goldstein et al., 2000; Broussard et al., 2001; Holzammer et al., 2001). No statistically significant difference was observed in viral loads of mandrills in different cohorts. Moreover, viral loads observed during the acute phase of SIVmnd-1 infection had no differences with respect to the infectious dose (Onanga et al., 2002; Pandrea et al., 2002).
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Table 1 SIVmnd-1 plasma viral load quantification (QC-RT-PCR) and antibody titers in experimentally infected, chronic naturally infected, and wild mandrills Experimentally infected captive mandrills
Day PIa
Copies/ml
anti-V3
anti-gp41
12A4
D180 D360 D540 D180 D360 D540 D180 D360 D540 D180 D360 D540 D180 D180 D180 D180 D180 D150 D180
334,000 93,750 93,750 150,000 93,750 93,750 93,750 66,964 93,750 66,964 66,964 18,750 66,964 155,000 66,964 40,000 334,821 334,821 334,821
800 1600 4800 400 1600 4800 400 1600 4800 1600 4800 14,400 1600 800 1600 200 400 1600 800
400 800 800 400 800 800 400 800 800 400 1600 3200 400 450 400 200 450 450 800
16E
2C2
10G
12C2 5I 5J 12A3 12A7 6B 16C Naturally infected captive mandrills
Year
Copies/ml
anti-V3
anti-gp41
17B
1992 1993 1995 1999 2000 1993 1999 2000 2000 2000
93,750 93,750 93,750 ND 93,750 ND 93,750 93,750 289,235 892,338
4800 4800 4800 4800 4800 0 400 1600 1600 800
6400 6400 12,800 6400 6400 200 800 1600 800 200
17D
17D2 17G Wild mandrills
Year
Copies/ml
anti-V3
anti-gp41
MS22-Lope MS23-Lope MS25-Lope MS21-Lope
2001 2001 2001 2001
334,821 206,596 66,964 66,964
1600 1600 1600 800
1600 400 800 400
a
P.I. ⫽ postinoculation.
Viral loads in captive naturally infected mandrills Serial sampling during a 15-year seroepidemiological follow-up of mandrills in the semifree ranging CIRMF colony has shown a cluster of SIVmnd-1 infections in a founder female mandrill F17 (GB1), three of her offspring (F17B, F17D, and M17G), and two of the F17D offspring (17D1 and 17D2). Dynamics of plasma SIVmnd-1 load in the founder female F17 (GB1) over a 15-year period have been published elsewhere (Pandrea et al., 2001). Here we present the dynamics of SIVmnd-1 plasma load on serial samples originating from four mandrills. F17B was born in 1987 and five sequential samples were tested (Table 1). QC-RT-PCR showed a remarkably narrow range of viral loads (93,750 copies/ml) in all but one sample. The remaining sample was stored at ⫺20°C and was used for antibody
testing. Both anti-V3 and anti-gp36 antibody titers were stable during the follow-up: 1/4800 and 1/6400, respectively. F17D was born in 1992 and three samples were tested. SIVmnd-1 viral loads for mandrill F17D were similar to F17B (93,750 copies/ml). However, antibody titers for F17D were significantly lower than for 17B (Table 1). Only one plasma sample was tested from the remaining animals. Mandrill 17D2 was born in 1997 and had been sampled once in 2000. Its viral load was significantly higher than observed in other mandrills (289,235 copies/ml) and antibody titers were significantly lower than those of 17B, but similar to those of 17D. The remaining mandrill (M17G) was born in 1996. His plasma viral load from 2000 was the highest in this study group (892,338 copies/ml). AntiSIVmnd-1 antibody titers were low in this mandrill as well (Table 1).
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Feral mandrills Previous reports of SIVmnd prevalence in mandrills were limited to SIVmnd-2 infections, which has a larger geographical distribution (Takehisha et al., 2001; Peeters et al., 2002). However, recent data originating from ecological and seroepidemiological studies have shown that SIVmnd-1 is readily transmitted in the wild, as 10 of 14 mandrills sampled in the Lope reserve in Gabon harbored SIVmnd-1 (Souquiere et al., 2001). In 2001, seven wild mandrills from the Lope reserve were sampled. Four were positive for SIVmnd-1 RNA. The two major aims of viral quantification in these mandrills were (1) to compare with results observed in the CIRMF colony in both naturally and experimentally infected mandrills, and (2) to determine if a high SIVmnd-1 prevalence in feral mandrills correlated with high viral loads. The results are presented in Table 1. All four wild mandrills harbored SIVmnd-1 viral loads in the same range as the captive mandrills (7 ⫻ 104–3 ⫻ 105 copies/ml), and also in the same range as African green monkeys and sooty mangabeys (Broussard et al., 2001; Goldstein et al., 2000; Holzammer et al., 2001; Chrakrabarti et al., 2000a; Silvestri et al., 2003). These results indicate that our primers were suitable for SIVmnd-1 quantification, as divergent viruses were amplified and viral loads were similar to experimentally inoculated animals. Flow cytometry Lymphocyte subsets (expressed as median and 25–75th percentile) were investigated in eight of the experimentally infected mandrills. Twelve uninfected mandrills served as controls. These mandrills were either age-matched (n ⫽ 6) or old mandrills, aged over 18 years (range 18 –23 years) (n ⫽ 6). This last group was examined to determine if an age-related decline of CD4 cells occurs in mandrills, similar to that reported in SIVsm-uninfected sooty mangabeys (Chakrabarti et al., 2000a, 2000b) and humans (Mo et al., 2003). When the haematological parameters (expressed as median and 25–75th percentile) of chronically infected (n ⫽ 8, mean age 8.6 years, range 3–19 years) and uninfected (n ⫽ 6) age-matched (mean age 8.2 years, range 4 –12) monkeys were analyzed by the Mann–Whitney test, no significant difference was noted in absolute lymphocyte counts between the two groups, expressed as ⫻103 cells/l blood (5.2 ⫾ 1.6 versus 4.63 ⫾ 2.32, in SIVmnd-1-infected and noninfected mandrills, respectively). However, the absolute CD20⫹, CD3⫹, and CD8⫹ counts were higher in the infected animals. Higher absolute CD8⫹ T cell numbers were found in the infected monkeys compared to age-matched uninfected mandrills (2230 ⫾ 1310 cells/l blood versus 1871 ⫾ 1160 cells/l blood, P ⬎ 0.05). Similar data were observed in relative values of CD8⫹ cells, which were higher in infected mandrills (mean: 52.39%, range: 39.4 – 62.5%) compared to
Fig. 1. (a) Box-plots of the relative numbers of lymphocyte subsets in mandrills. The plots correspond to young age-matched uninfected mandrills (mean 7.9 years, range 6 –11 years) (䊐); old uninfected mandrills (mean 18.6 years; range: 17–22 years) ( ); and SIVmnd-1-infected mandrills (mean 7.4 years; range 3–12 years) (■). (b) Activation status of CD4⫹ and CD8⫹ cells in young age-matched SIVmnd-1 uninfected (䊐 and infected (■) mandrills, as determined by HLA-DR and/or CD28 coexpression. In the box plots representation, the box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the dots extend down to the smallest value and up to the largest value.
young age-matched uninfected mandrills (mean: 45.63%; range: 35.3–58.2%) (Fig. 1). The effect of age was expressed by a slight decrease in total lymphocyte in old uninfected controls. Thus, mean absolute lymphocyte counts, expressed as ⫻103 cells/l blood, were 3.92 ⫾ 1.2 in old uninfected mandrills, which was similar to that previously reported in old sooty mangabeys (Chakrabarti et al., 2000b). Mean percentages of CD8⫹ cells in old uninfected mandrills was 40.1% (range: 30.1 ⫾ 51.7%). CD3⫹ cells were also higher in SIVmnd-1-infected mandrills (Fig. 1a), but this difference was not statistically significant between the three groups. Differences were observed in the expression of activation markers when two groups of animals, SIVmnd-1-infected versus uninfected, were compared (Fig. 1b). CD8⫹
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HLA-DR⫹ cells were increased in infected mandrills (mean: 7%, range: 6.1–10.2) compared to age-matched uninfected controls (mean: 5.51%, range: 3.2–7.1). CD8⫹ CD28⫹ cells were also higher in infected mandrills (mean: 17.81%, range: 14.5–21.5%) compared to uninfected controls (mean: 15.55%, range: 13.2–18.7%). No difference in activation markers were detected on CD4⫹ cells between infected and uninfected mandrills (Fig. 1b). In summary, minor differences were detected between infected and uninfected mandrills, mostly within the CD3⫹ and CD8⫹ cells, and activation status in the latter. However, these differences were not statistically significant.
Discussion In this study the viral load in SIVmnd-1 chronically infected mandrills was investigated. Although previous reports measured viral loads in captive African monkeys (Goldstein et al., 2000; Broussard et al., 2001; Chakrabarti et al., 2000a; Ling et al., 2003; Silvestri et al., 2003), to our knowledge, this is the first report describing SIV viral loads in wild natural infected hosts. The only other study which investigated the viral burden in wild sooty mangabeys measured only cellular proviral loads and not plasma RNA (Chen et al., 1996). No significant differences were observed in viral loads of mandrills from experimentally and naturally infected study groups. Chronic viral loads were also similar in animals receiving different infectious doses of SIVmnd-1, showing that dose had no detectable influence on the dynamics of acute (Onanga et al., 2002; Pandrea et al., 2002) or chronic SIVmnd-1 infection. A potential limitation of our study as a model of the natural history of the SIVmnd-1 infections is that all experimentally infected animals received the same viral strain (SIVmnd-1 GB1). However, this is also true of SIV pathogenesis studies in rhesus macaques, which are infected with very similar SIVmac strains (Mansfield et al., 1995). Our results showed that experimental infection reproduced similar patterns of virushost dynamics as observed in naturally infected mandrills. Viral loads described here in wild mandrills were in the same range as those reported by others examining sooty mangabeys and AGMs (Rey-Cuille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Ling et al., 2003; Silvestri et al., 2003). The fact that viral loads are the same between wild and captive mandrills indicates that diet, medical care, and other factors play little role in determining viral load. Conversely, these latter factors plus a lack of natural predators and not a lower viral replication are likely to be responsible for the longer life span of captive monkeys compared to wild ones. Although the sequences of SIVmnd-1 strains from wild mandrills have been not analyzed in this study, previous analysis has shown significant viral diversity in SIVmnd-1infected mandrills from the Lope reserve (Souquiere et al., 2001). However, our primers were designed from a highly
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conserved region of the SIVmnd-1 genome and therefore are likely to correctly amplify divergent SIVmnd-1 strains. In humans, HIV-1 viral diversity may significantly influence the performances of different commercially available viral load quantification kits. Thus, bDNA designed to map the polymerase region has a better sensitivity than gagbased Monitor–Roche for the quantification of divergent HIV-1 group M viruses, which may vary in up to 20% of pol sequences (Damond et al., 1999). Our strategy of amplifying the integrase region seems more appropriate for the quantification of divergent viruses than a gag-based strategy. However, in a recent study of viral loads in sooty mangabeys from the Yerkes National Primate Research Centre, gag quantification gave similar results with a UTRbased method for the quantification of SIVsm [Silvestri et al., 2003), despite the cocirculation in the Yerkes colony of four different SIVsm lineages (Ling et al., 2003; A. Kaur, C. Apetrei, and P.A. Marx, unpublished observation). It is not clear why the prevalence of SIVmnd-1 in mandrils from the Lope reserve is higher than generally reported for other natural SIV infection (Jolly et al., 1996, Peeters et al., 2002). Since no differences were detected in viral loads between wild and captive mandrills, the differences in prevalence between different groups of the same species or different nonhuman primate species may not be related to virological or host factors but rather to behavior which favors viral transmission (Santiago et al., 2003). SIV might be concentrated in specific nonhuman primate populations rather than widely distributed. This is also consistent with the hypothesis that lentivirus transmission to humans only occurred in relatively limited areas in Central and West Africa (Peeters et al., 2002). In humans and macaques there is a strong correlation between the pattern of viral replication and the clinical course of infection, a high viral load often predicting early disease (Mellors et al., 1997; Lifson et al., 1997). Previous reports suggested that the lack of pathogenicity of nonhuman primate lentiviruses in their natural hosts might be due to low viral replication (Hartung et al., 1992). However, several subsequent studies as well as this study have shown that during the chronic phase of infection, primate lentiviruses replicate at moderate to high levels without clinical or pathological signs (Rey-Couille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Holzammer et al., 2001). Therefore the lack of pathogenicity of SIVs, including SIVmnd-1, cannot be explained by limited replication in their natural host. Anti-gp36 and anti-V3 antibody titers were performed to evaluate antibody responses and relationship to virus load. The immunodominant region of the gp36 is one of the most conserved SIV epitopes and it is useful in diagnostic tests in both humans and nonhuman primates. Detecting anti-gp36 antibodies is a very effective diagnostic tool (Simon et al., 2001). Although in HIV-1 infection V3 region of gp120 is highly variable, this region is significantly more conserved in SIV infection (Franchini et al., 1990). This is supported
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by the finding that all the infected mandrills tested so far reacted with GB-1 V3 peptide. Our SIVmnd-1 V3 peptide is able to even detect antibodies in SIVmnd-2-infected mandrills or SIVdrl-infected drills (Souquiere et al., 2001; Simon et al., 2001). This is not surprising, as SIVmnd-2 and SIVdrl are recombinant viruses with a SIVmnd-1 envelope (Souquiere et al., 2001; Hu et al., 2003). The anti-V3 antibodies were tested because a decrease in titers is often correlated with disease progression and a decline in serum neutralizing activity in HIV-1-infected humans (Fenouillet et al., 1995). We have also reported a loss of anti-V3 antibodies in an SIVmnd-1-infected mandrill with immune suppression (Pandrea et al., 2001). However, none of the mandrills presented here showed decreased anti-V3 titers. Also, there was no clear correlation between viral load and anti-V3 titers. Theoretically, a more meaningful gauge of immune function and its relationship to viral load may have been neutralizing antibody responses. However, SIVmnd-1 GB1 grows poorly in cell culture, and this has hampered a proper evaluation of neutralizing antibody responses. Also, it should be noted that in other SIV naturally infected African monkeys, neutralizing antibody responses are generally low (Apetrei et al., 2004). From a clinical point of view, African monkeys may be similar to long-term nonprogressing HIV-infected humans. It was suggested that SIV adaptation to the nonprogressor status may be due to induction of a persistent infection with a symptom-free period exceeding the normal life span of most monkeys (Pandrea et al., 2001). However, in African nonhuman primates infected with SIV, the pathologic mechanisms must be different from those observed in HIV1-infected long-term nonprogressors, in whom the low HIV-1 loads are due to active cellular immune responses (Cao et al., 1995). In contrast, African monkeys have high viral loads which are associated with an attenuated antiviral cellular immune response (Chakrabarti et al., 2000a; Holznagel et al., 2002; Silvestri et al., 2003). Moreover, this equilibrium is maintained for long periods of time, without any clinical and immunological sign of immune suppression. Thus, our analysis of immunophenotypic markers in SIVmnd-1-infected mandrills was designed to determine if high levels of viremia have any effect on T cells. None of the parameters included in our immunophenotypic analysis showed significant differences between SIVmnd-1-infected and uninfected mandrills. Chronically infected mandrills were found to maintain relatively normal CD4⫹ counts in peripheral blood, despite years of infection. The slight increase in levels of CD8⫹ HLA-DR⫹ cells observed in chronically infected mandrills was not associated with decreased levels of CD8⫹ CD28⫹ cells, unlike results observed in humans (Giorgi et al., 1999; Sousa et al., 2002). However, this data are similar to data previously reported in SIVsm-infected sooty mangabeys (Silvestri et al., 2003). Our results indicate that SIVmnd-1 is well adapted to its mandrill host, and that cross-species transmission of lentiviruses in the wild can be controlled by the new primate
host. The mechanism(s) of this resistance to SIV of African monkeys should be further investigated to gain insights to control HIV infection.
Materials and methods Animals A total of 19 SIVmnd-1 chronically infected mandrills were examined, 4 of which resided in a large semi-free enclosure, and were naturally infected in captivity. Furthermore, these four (17B, 17D, 17D2, 17G) were offspring of the founder SIVmnd-1 GB1 female with whom they were raised/housed, suggesting that vertical transmission occurred. However, serial plasma samples were only examined on two of these mandrills: F17B (1992, 1993, 1995, 1999, and 2000) and F17D (1993, 1999, and 2000). Eleven mandrills were experimentally infected in two different experiments: mandrills 16E, 2C2, 12A4, 10G, 12C2 were intravenously infected with 3000 TCID50. Data on acute infection in these animals have been published elsewhere (Onanga et al., 2002). Mandrills 12A3, 16C, 12A7, 5I, 5J, 6B were infected intravenously with 300 TCID50 SIVmnd-1 and viral loads were measured (day 180). In both experiments, the inoculum was derived from plasma prepared as previously described (Onanga et al., 2002). An additional 11 uninfected mandrills were used as controls for flow cytometry analyses. In the study group, 5 animals were male (2C2, 12A4, 17G, 12C2, 10G) and 10 were female (17B, 17D, 17D2, 12A3, 16C, 16E, 12A7, 5I, 5J, 6B). The mean age of the animals was 8.6 (range 3–19). The mean weight for the males was 30 kg and 15 kg for females. Mandrill 12C2 died in 2000 of gastric dilatation, with no evidence of AIDS (Onanga et al., 2002). The wild mandrills originated from the Lope reserve in Central Gabon. All were adult solitary males in good health at the time of capture. No follow-up was possible for these mandrills because they were released. Sampling Animals were anesthetized with ketamine–HCl and 7–10 ml of whole blood was collected in EDTA. Plasma was separated and stored at ⫺80°C until used for semiquantitative viral load analysis and serology. Whole blood was used for flow cytometry as described below. Quantification of viral RNA in plasma RNA quantification was performed by a limiting-dilution RT-PCR assay specific for SIVmnd-1 (Onanaga et al., 2002). The primers were MP5 (5⬘-CCA GAT AAG TGG AAG ATA GAA AAG-3⬘) and MP6 (5⬘-GGG AGA TAT GGG AAG ATT GGG-3⬘), allowing the amplification of a 476-bp region within the SIVmnd-1 GB1 pol gene as pre-
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viously described (Nerrienet et al., 1998). Since the virus used in this study was derived from the SIVmnd-1 GB1, the possibility of mismatches was minimal. RNA was extracted from plasma using the QIAamp Viral RNA Mini kit (Qiagen, Courtaboeuf, France). The RNA extracted from 540 l of plasma was subjected to seven 5- to 10-fold serial dilutions which were used for the RT-PCR (one step RT-PCR kit, Qiagen). Reverse transcription was performed at 50°C for 30 min in the presence of MP5 and MP6, followed by 47 cycles of 95°C for 15 s, 55°C for 45 s, and 72°C for 1 min, with a final extension of 7 min at 72°C. The absolute target copy numbers were determined by amplification in parallel of known amounts of a SIVmnd-1 RNA external standard. The standard was obtained by amplifying the 476-bp region of SIVmnd-1 GB1 pol from F17 genomic DNA. The PCR product was cloned into the pCR 2.1 vector using the topoTA Cloning Kit (Invitrogen, Groningen, The Netherlands). The 5⬘-3⬘ sense orientation of the insert was verified by sequence analyses. A clone containing the insert in 5⬘-3⬘ orientation was chosen and subjected to PCR using the primer pair pCRT7S 5⬘ (GCG TAA TAC GAC TCA CTA TAG GGA GAG GA GCTC GAG CGG CCG CCA GTG TG) and pCRT7AS (5⬘-ATT ACG CCA AGC TTG GTA CCG AG3⬘), which hybridize to regions just upstream and downstream of the pCR2.1 plasmid polylinker sequence. Cycling conditions for this PCR consisted of an initial DNA denaturation step, followed by 40 cycles that included a denaturation step at 92°C for 30 s, primer annealing at 62°C for 45 s, and DNA synthesis at 72°C for 45 s, with a final extension of 7 min at 72°C. The resulting PCR product contained the cloned insert together with a T7 promoter sequence at its 5⬘ end. After clean up of the PCR product (PCR purification kit, Qiagen, Courtaboeuf, France) this DNA was used as a template for an in vitro transcription (MEGAscript kit, Ambion, Austin, TX). The resulting RNA was DNase digested and nonincorporated nucleotides were eliminated using a Chroma Spin-30 DEPC H20 Column (Clontech, Palo Alto, CA). Size and purity of the synthesized RNA were verified on a denaturating-PAGE gel. The concentration of the in vitro transcript was determined by a highly sensitive and specific Gene Quant II spectrophotometric operation system as already described (Onanga et al., 2002). Aliquots were stored at ⫺80°C until use. Immediately before usage, one aliquot of RNA was thawed and diluted in RNase free H2O and five dilutions of the RNA were used as external standard in each experiment. The sensitivity of the assay was 2 ⫻ 102 RNA copies/ml of plasma.
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Table 2 Antibody panel used for evaluating host immune dynamics in mandrills chronically infected with SIVmnd-1 mAB-PEa (clone, source) d
IgG1 (BD ) IgG1 (BD) IgG1 (BD) CD4 (Leu-3a BD) CD8 (Leu-2a BD) CD28 (Leu-28BD) CD28 (Leu-28BD) CD4 (Leu-3a BD) CD4 (Leu-3a BD) CD8 (Leu-2a BD)
mAB-FITCb (clone, source)
Cell type defined
IgG1 (PN0639 ITc) Control Control IgG1 (IT) Control IgG2a (BD) Control CD20 (Leu-16 BD) B cells CD3 (FN-18 BSe) T cells, T helper cells CD3 (FN-18 BS) T cells, T cytotoxic cells CD4 (Leu-3 BD) Costimulatory T helper cells CD8 (Leu 2a BD) Costimulatory T cytotoxic cells CD8 (Leu 2a BD) T helper, T cytotoxic cells HLA-DR (BD) Activated MHC II CD4⫹ cells HLA-DR (BD) Activated MHC II CD8⫹ cells
a
Phycoerythrin (PE). Fluorescein isothiocyanate (FITC). c ImmunoTech (IT). d BioSource (BS). e Becton Dickinson (BD). b
tested against gp36 and V3 peptide, respectively. The cutoff was established at 0.1 o.d. Flow cytometry analysis To quantify lymphocyte subsets and examine lymphocyte activation in infected versus uninfected animals, a variety of antibodies against cell-surface markers were used, as summarized in Table 2. Samples from noninfected (M5H, M2I, F12A3, F5J, F16C, F12A7, and F5I), and infected (F17, F17D, M2C2, M12A4, and M10G) mandrills were analyzed by flow cytometry. One hundred microliters of EDTA-treated whole blood was incubated in the dark with 5 l antibody for 30 min at 4°C. Red blood cells were lysed using a standard ammonium chloride lysing solution. Cells were then washed twice in phosphate-buffered saline (PBS) and resuspended in PBS with 1% paraformaldehyde. Data were acquired using a FACsCalibur flow cytometer (Immunocytometry System, Becton Dickinson) and analyzed with Cell Quest software (Immunocytometry System, Becton Dickinson). Statistical analysis and data presentation Flow cytometriy data were analyzed for significant differences (P ⬍ 0.05) using the Mann–Whitney test. Correlations were calculated and expressed as the Spearman coefficient of correlation (rs). Data were displayed using the box-and-whisker plot method (Fig. 1).
Primate immunodeficiency viruses ELISA (PIV-ELISA) Acknowledgments Antibody titers to both gp36 and V3 peptide were measured using a PIV-ELISA, as previous described (Simon et al., 2001). Twofold serial dilutions from 1/100 to 1/12,800 and from 1/100 to 1/43,200 were done for each sample
This work was supported by grants from Agence Nationale pour la Recherche sur le SIDA (ANRS) and NIH (RO1-AI44596).
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Virology 317 (2003) 119 –127
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High levels of SIVmnd-1 replication in chronically infected Mandrillus sphinx Ivona Pandrea,a,b,1 Richard Onanga,a,1 Christopher Kornfeld,c Pierre Rouquet,a Olivier Bourry,a Stephen Clifford,a Paul T. Telfer,a Kate Abernethy,a Lee TW White,a Paul Ngari,a Michaela Mu¨ller-Trutwin,c Pierre Roques,a,d Preston A. Marx,b Francois Simon,a,e and Cristian Apetreia,b,* a
Laboratoire de Virologie, UGENET, SEGC, Re´serve de la Lope´, Centre de Primatologie, Centre International de Recherches Me´dicales, BP769, Franceville, Gabon b Tulane National Primate Research Center, Covington, LA 70433, USA c Unite´ de Biologie des Re´trovirus, Institut Pasteur, 29, Rue Dr. Roux, 75724 Paris Cedex 15, France d Service de Neurovirologie, Commisariod a` l’Energze Atomygue, 92265 Foulenay aux Roses, France e Hopital Charles Nicolle, 76031 Rouen, France Received 14 May 2003; returned to author for revision 17 June 2003; accepted 14 August 2003
Abstract Viral loads were investigated in SIVmnd-1 chronically infected mandrills and the results were compared with those previously observed in other nonpathogenic natural SIV infections. Four naturally and 11 experimentally SIVmnd-1-infected mandrills from a semi-free-ranging colony were studied during the chronic phase of infection. Four SIVmnd-1-infected wild mandrills were also included for comparison. Twelve uninfected mandrills were used as controls. Viral loads in all chronically infected mandrills ranged from 105 to 9 ⫻ 105 copies/ml and antibody titers ranged from 200 to 14,400 and 200 to 12,800 for anti-V3 and anti-gp36, respectively. There were no differences between groups of wild and captive mandrills. Both parameters were stable during the follow-up, and no clinical signs of immune suppression were observed. Chronic SIVmnd-1-infected mandrills presented slight increases in CD20⫹ and CD28⫹/CD8⫹ cell counts, and a slight decrease in CD4⫹/CD3⫹ cell counts. A slight CD4⫹/CD3⫹ cell depletion was also observed in old uninfected controls. Similar to other nonpathogenic models of lentiviral infection, these results show a persistent high level of SIVmnd-1 replication during chronic infection of mandrills, with minimal effects on T cell subpopulations. © 2003 Elsevier Inc. All rights reserved. Keywords: SIVmnd-1; Pathogenesis; Mandrills; Viral load; Lymphocyte subsets
Introduction More than 30 simian immunodeficiency viruses (SIVs) naturally infect nonhuman African primates (Apetrei et al., 2004). Their overall seroprevalence in the wild is very high (estimated 18 –30%) (Jolly et al., 1996; Chen et al., 1996; Souquiere et al., 2001; Peeters et al., 2002). Although they were named SIV because of their structural and biological
* Corresponding author. Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433. Fax: ⫹1-985-871-6248. E-mail address: [email protected] (C. Apetrei). 1 These authors contributed equally to this study. 0042-6822/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2003.08.015
resemblance to the human immunodeficiency virus (HIV), SIV infections are mostly nonpathogenic in their natural hosts (Beer et al., 1998; Rey-Cuille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Silvestri et al., 2003). A few exceptions have been documented in mandrills and African green monkeys (Pandrea et al., 2001; Souquiere et al., 2001; Traina-Dorge et al., 1992). Disease is probably a rare consequence because of long-term adaptation of SIV to its natural African primate hosts, which is reflected in continuous and sustained viral replication with minimal effects on immune function (Kaur et al., 1998; Silvestri et al., 2003). There is evidence showing that SIVs have pathogenic
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potential. Cross-species transmission of SIVsm to macaques results in increased pathogenicity and induction of simian acquired immunedeficiency syndrome (sAIDS) (Letvin et al., 1985; Murphey-Corb et al., 1986). HIV-1 and HIV-2, which have simian origins, are responsible for the AIDS pandemic (Peeters et al., 1989; Gao et al., 1999; Chen et al., 1996). Experimental transmission of SIVagm from African green monkeys (AGMs) and SIVIhoest from I’hoesti monkeys to pig-tailed macaques has also shown pathogenic potential and immunosuppression (Hirsch et al., 1995, 1999). In these cases, high viral loads induce massive activation and destruction of the immune system (Alimonti et al., 2003). Therefore, plasma viral load is considered the best predictor of the clinical outcome in humans and macaques (Mellors et al., 1996; Lifson et al., 1997). In this study, we have investigated the relationship between SIV and its natural host in a new model, SIVmnd-1 chronic infection of mandrills. The probable origin of SIVmnd-1 was reported to be from Cercopithecus lhoesti solatus to mandrills (Hirsch et al., 1999; Beer et al., 1999). This observation raised the question as to whether or not mandrills should be considered as long-term natural hosts of SIVmnd-1 and whether the course of infection would be the same as in other nonpathogenic infections. The preponderance of evidence indicates that mandrills are a natural host because recent studies have shown that SIVmnd-1 is highly prevalent in wild mandrills, suggesting that cross-species transmission was an ancient event and that the virus infects this species naturally (Souquiere et al., 2001). SIVmnd-1 replicates to high levels during experimental primary infection in mandrills and this replication has only slight and transitory effects on the dynamics of immune cells (Onanga et al., 2002). This pattern is very similar to those observed during the primary infection of other nonhuman primate species (Diop et al., 2000; Kaur et al., 1998). Here we address the hypothesis as to whether or not mandrills represent an intermediate level between pathogenic and nonpathogenic SIV infections or whether or not the virus is adapted to the mandrill host. Only one SIVmnd-1 strain (GB-1) is available and it was adapted in vitro and reported to use CXCR4 as the main coreceptor, which is highly unusual for SIV (Schols and De Clerq, 1998; Beer et al., 2001). Therefore, SIVmnd-1 (GB-1) may have a higher pathogenic potential compared to SIVs which use CCR-5 as the major coreceptor (Marx and Chen, 1998). However, SIVmnd-1 GB1 recovered during the primary infection used CCR5 and Bonzo and not CXCR4 (R. Onanga, unpublished observation). As chronic infection reflects the long-term natural history of SIV, the aim of the present study was to measure the dynamics of RNA plasma viral load, anti-SIVmnd-1 antibody titers, and lymphocyte subsets in mandrills chronically infected with SIVmnd-1 to compare these data with those previously observed in other nonpathogenic models. Data from wild mandrills are also presented for comparison.
Results Clinical and immunological follow-up of chronically infected SIVmnd-1 mandrills No clinical signs of AIDS were observed during annual follow-ups of chronically, naturally infected mandrills (17B, 17D, 17G, 17D2) in captivity. Eleven experimentally infected animals (n ⫽ 11) were examined every 3 months. No follow-up was possible for the wild mandrills, but all were clinically normal at the time of sampling. Viral load quantification in experimentally infected mandrills In HIV-infected humans and SIV-infected rhesus macaques the quantification of plasma viral RNA provides a measure of the magnitude of virus replication (Mellors et al., 1997; Lifson et al., 1997). For viral RNA loads in plasma, a QC-RT-PCR assay specific for SIVmnd-1 was developed (Onanga et al., 2002) using primers MP5/MP6 from a conserved region in integrase of SIVmnd-1 GB-1 (Nerrienet et al., 1998). There were no mismatches (Onanga et al., 2002). Our previous studies on the acute phase of SIVmnd-1 infection showed that high viral loads obtained by QC-RT-PCR directly correlated with both p24 Ag titers and plasma RT activity (Onanga et al., 2002). Experimentally infected mandrills included in this study were mandrills from a primary infection experiment (12A4, 2C2, 10G, 12C2) inoculated intravenously (iv) with 3000 TCID50, and dams in a breastfeeding transmission experiment (5I, 5J, 12A3, 12A7, 6B, 16C), which received 300 TCID50 iv of the same stock. Mandrill 16E received 10 ml of blood from the mandrill naturally infected with GB-1 (F17). This virus was titered at 4 TCID50 on SupT1 cells, a value which corresponded to 896,000 RNA copies/ml (Onanga et al., 2002). In experimentally infected mandrills, the viral set point was reached between days 30 and 60 postchallenge (Onanga et al., 2002). In this present study we quantified the SIVmnd-1 RNA loads in 19 samples collected between days 180 and 540 postchallenge from all 11 experimentally infected mandrills (Table 1). SIVmnd-1 plasma viral loads are presented in Table 1. Although results obtained at days 180 and 360 for mandrills 12A4, 16E, 2C2, and 10G have been presented elsewhere, these points are also showed in Table 1 for comparison. Viral RNA levels ranged from 18,000 to 334,000 copies/ml, similar to the range observed in SIVsm-infected sooty mangabeys (ReyCouille et al., 1998; Ling et al., 2003; Silvestri et al., 2003) and AGMs (Goldstein et al., 2000; Broussard et al., 2001; Holzammer et al., 2001). No statistically significant difference was observed in viral loads of mandrills in different cohorts. Moreover, viral loads observed during the acute phase of SIVmnd-1 infection had no differences with respect to the infectious dose (Onanga et al., 2002; Pandrea et al., 2002).
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Table 1 SIVmnd-1 plasma viral load quantification (QC-RT-PCR) and antibody titers in experimentally infected, chronic naturally infected, and wild mandrills Experimentally infected captive mandrills
Day PIa
Copies/ml
anti-V3
anti-gp41
12A4
D180 D360 D540 D180 D360 D540 D180 D360 D540 D180 D360 D540 D180 D180 D180 D180 D180 D150 D180
334,000 93,750 93,750 150,000 93,750 93,750 93,750 66,964 93,750 66,964 66,964 18,750 66,964 155,000 66,964 40,000 334,821 334,821 334,821
800 1600 4800 400 1600 4800 400 1600 4800 1600 4800 14,400 1600 800 1600 200 400 1600 800
400 800 800 400 800 800 400 800 800 400 1600 3200 400 450 400 200 450 450 800
16E
2C2
10G
12C2 5I 5J 12A3 12A7 6B 16C Naturally infected captive mandrills
Year
Copies/ml
anti-V3
anti-gp41
17B
1992 1993 1995 1999 2000 1993 1999 2000 2000 2000
93,750 93,750 93,750 ND 93,750 ND 93,750 93,750 289,235 892,338
4800 4800 4800 4800 4800 0 400 1600 1600 800
6400 6400 12,800 6400 6400 200 800 1600 800 200
17D
17D2 17G Wild mandrills
Year
Copies/ml
anti-V3
anti-gp41
MS22-Lope MS23-Lope MS25-Lope MS21-Lope
2001 2001 2001 2001
334,821 206,596 66,964 66,964
1600 1600 1600 800
1600 400 800 400
a
P.I. ⫽ postinoculation.
Viral loads in captive naturally infected mandrills Serial sampling during a 15-year seroepidemiological follow-up of mandrills in the semifree ranging CIRMF colony has shown a cluster of SIVmnd-1 infections in a founder female mandrill F17 (GB1), three of her offspring (F17B, F17D, and M17G), and two of the F17D offspring (17D1 and 17D2). Dynamics of plasma SIVmnd-1 load in the founder female F17 (GB1) over a 15-year period have been published elsewhere (Pandrea et al., 2001). Here we present the dynamics of SIVmnd-1 plasma load on serial samples originating from four mandrills. F17B was born in 1987 and five sequential samples were tested (Table 1). QC-RT-PCR showed a remarkably narrow range of viral loads (93,750 copies/ml) in all but one sample. The remaining sample was stored at ⫺20°C and was used for antibody
testing. Both anti-V3 and anti-gp36 antibody titers were stable during the follow-up: 1/4800 and 1/6400, respectively. F17D was born in 1992 and three samples were tested. SIVmnd-1 viral loads for mandrill F17D were similar to F17B (93,750 copies/ml). However, antibody titers for F17D were significantly lower than for 17B (Table 1). Only one plasma sample was tested from the remaining animals. Mandrill 17D2 was born in 1997 and had been sampled once in 2000. Its viral load was significantly higher than observed in other mandrills (289,235 copies/ml) and antibody titers were significantly lower than those of 17B, but similar to those of 17D. The remaining mandrill (M17G) was born in 1996. His plasma viral load from 2000 was the highest in this study group (892,338 copies/ml). AntiSIVmnd-1 antibody titers were low in this mandrill as well (Table 1).
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Feral mandrills Previous reports of SIVmnd prevalence in mandrills were limited to SIVmnd-2 infections, which has a larger geographical distribution (Takehisha et al., 2001; Peeters et al., 2002). However, recent data originating from ecological and seroepidemiological studies have shown that SIVmnd-1 is readily transmitted in the wild, as 10 of 14 mandrills sampled in the Lope reserve in Gabon harbored SIVmnd-1 (Souquiere et al., 2001). In 2001, seven wild mandrills from the Lope reserve were sampled. Four were positive for SIVmnd-1 RNA. The two major aims of viral quantification in these mandrills were (1) to compare with results observed in the CIRMF colony in both naturally and experimentally infected mandrills, and (2) to determine if a high SIVmnd-1 prevalence in feral mandrills correlated with high viral loads. The results are presented in Table 1. All four wild mandrills harbored SIVmnd-1 viral loads in the same range as the captive mandrills (7 ⫻ 104–3 ⫻ 105 copies/ml), and also in the same range as African green monkeys and sooty mangabeys (Broussard et al., 2001; Goldstein et al., 2000; Holzammer et al., 2001; Chrakrabarti et al., 2000a; Silvestri et al., 2003). These results indicate that our primers were suitable for SIVmnd-1 quantification, as divergent viruses were amplified and viral loads were similar to experimentally inoculated animals. Flow cytometry Lymphocyte subsets (expressed as median and 25–75th percentile) were investigated in eight of the experimentally infected mandrills. Twelve uninfected mandrills served as controls. These mandrills were either age-matched (n ⫽ 6) or old mandrills, aged over 18 years (range 18 –23 years) (n ⫽ 6). This last group was examined to determine if an age-related decline of CD4 cells occurs in mandrills, similar to that reported in SIVsm-uninfected sooty mangabeys (Chakrabarti et al., 2000a, 2000b) and humans (Mo et al., 2003). When the haematological parameters (expressed as median and 25–75th percentile) of chronically infected (n ⫽ 8, mean age 8.6 years, range 3–19 years) and uninfected (n ⫽ 6) age-matched (mean age 8.2 years, range 4 –12) monkeys were analyzed by the Mann–Whitney test, no significant difference was noted in absolute lymphocyte counts between the two groups, expressed as ⫻103 cells/l blood (5.2 ⫾ 1.6 versus 4.63 ⫾ 2.32, in SIVmnd-1-infected and noninfected mandrills, respectively). However, the absolute CD20⫹, CD3⫹, and CD8⫹ counts were higher in the infected animals. Higher absolute CD8⫹ T cell numbers were found in the infected monkeys compared to age-matched uninfected mandrills (2230 ⫾ 1310 cells/l blood versus 1871 ⫾ 1160 cells/l blood, P ⬎ 0.05). Similar data were observed in relative values of CD8⫹ cells, which were higher in infected mandrills (mean: 52.39%, range: 39.4 – 62.5%) compared to
Fig. 1. (a) Box-plots of the relative numbers of lymphocyte subsets in mandrills. The plots correspond to young age-matched uninfected mandrills (mean 7.9 years, range 6 –11 years) (䊐); old uninfected mandrills (mean 18.6 years; range: 17–22 years) ( ); and SIVmnd-1-infected mandrills (mean 7.4 years; range 3–12 years) (■). (b) Activation status of CD4⫹ and CD8⫹ cells in young age-matched SIVmnd-1 uninfected (䊐 and infected (■) mandrills, as determined by HLA-DR and/or CD28 coexpression. In the box plots representation, the box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the dots extend down to the smallest value and up to the largest value.
young age-matched uninfected mandrills (mean: 45.63%; range: 35.3–58.2%) (Fig. 1). The effect of age was expressed by a slight decrease in total lymphocyte in old uninfected controls. Thus, mean absolute lymphocyte counts, expressed as ⫻103 cells/l blood, were 3.92 ⫾ 1.2 in old uninfected mandrills, which was similar to that previously reported in old sooty mangabeys (Chakrabarti et al., 2000b). Mean percentages of CD8⫹ cells in old uninfected mandrills was 40.1% (range: 30.1 ⫾ 51.7%). CD3⫹ cells were also higher in SIVmnd-1-infected mandrills (Fig. 1a), but this difference was not statistically significant between the three groups. Differences were observed in the expression of activation markers when two groups of animals, SIVmnd-1-infected versus uninfected, were compared (Fig. 1b). CD8⫹
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HLA-DR⫹ cells were increased in infected mandrills (mean: 7%, range: 6.1–10.2) compared to age-matched uninfected controls (mean: 5.51%, range: 3.2–7.1). CD8⫹ CD28⫹ cells were also higher in infected mandrills (mean: 17.81%, range: 14.5–21.5%) compared to uninfected controls (mean: 15.55%, range: 13.2–18.7%). No difference in activation markers were detected on CD4⫹ cells between infected and uninfected mandrills (Fig. 1b). In summary, minor differences were detected between infected and uninfected mandrills, mostly within the CD3⫹ and CD8⫹ cells, and activation status in the latter. However, these differences were not statistically significant.
Discussion In this study the viral load in SIVmnd-1 chronically infected mandrills was investigated. Although previous reports measured viral loads in captive African monkeys (Goldstein et al., 2000; Broussard et al., 2001; Chakrabarti et al., 2000a; Ling et al., 2003; Silvestri et al., 2003), to our knowledge, this is the first report describing SIV viral loads in wild natural infected hosts. The only other study which investigated the viral burden in wild sooty mangabeys measured only cellular proviral loads and not plasma RNA (Chen et al., 1996). No significant differences were observed in viral loads of mandrills from experimentally and naturally infected study groups. Chronic viral loads were also similar in animals receiving different infectious doses of SIVmnd-1, showing that dose had no detectable influence on the dynamics of acute (Onanga et al., 2002; Pandrea et al., 2002) or chronic SIVmnd-1 infection. A potential limitation of our study as a model of the natural history of the SIVmnd-1 infections is that all experimentally infected animals received the same viral strain (SIVmnd-1 GB1). However, this is also true of SIV pathogenesis studies in rhesus macaques, which are infected with very similar SIVmac strains (Mansfield et al., 1995). Our results showed that experimental infection reproduced similar patterns of virushost dynamics as observed in naturally infected mandrills. Viral loads described here in wild mandrills were in the same range as those reported by others examining sooty mangabeys and AGMs (Rey-Cuille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Ling et al., 2003; Silvestri et al., 2003). The fact that viral loads are the same between wild and captive mandrills indicates that diet, medical care, and other factors play little role in determining viral load. Conversely, these latter factors plus a lack of natural predators and not a lower viral replication are likely to be responsible for the longer life span of captive monkeys compared to wild ones. Although the sequences of SIVmnd-1 strains from wild mandrills have been not analyzed in this study, previous analysis has shown significant viral diversity in SIVmnd-1infected mandrills from the Lope reserve (Souquiere et al., 2001). However, our primers were designed from a highly
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conserved region of the SIVmnd-1 genome and therefore are likely to correctly amplify divergent SIVmnd-1 strains. In humans, HIV-1 viral diversity may significantly influence the performances of different commercially available viral load quantification kits. Thus, bDNA designed to map the polymerase region has a better sensitivity than gagbased Monitor–Roche for the quantification of divergent HIV-1 group M viruses, which may vary in up to 20% of pol sequences (Damond et al., 1999). Our strategy of amplifying the integrase region seems more appropriate for the quantification of divergent viruses than a gag-based strategy. However, in a recent study of viral loads in sooty mangabeys from the Yerkes National Primate Research Centre, gag quantification gave similar results with a UTRbased method for the quantification of SIVsm [Silvestri et al., 2003), despite the cocirculation in the Yerkes colony of four different SIVsm lineages (Ling et al., 2003; A. Kaur, C. Apetrei, and P.A. Marx, unpublished observation). It is not clear why the prevalence of SIVmnd-1 in mandrils from the Lope reserve is higher than generally reported for other natural SIV infection (Jolly et al., 1996, Peeters et al., 2002). Since no differences were detected in viral loads between wild and captive mandrills, the differences in prevalence between different groups of the same species or different nonhuman primate species may not be related to virological or host factors but rather to behavior which favors viral transmission (Santiago et al., 2003). SIV might be concentrated in specific nonhuman primate populations rather than widely distributed. This is also consistent with the hypothesis that lentivirus transmission to humans only occurred in relatively limited areas in Central and West Africa (Peeters et al., 2002). In humans and macaques there is a strong correlation between the pattern of viral replication and the clinical course of infection, a high viral load often predicting early disease (Mellors et al., 1997; Lifson et al., 1997). Previous reports suggested that the lack of pathogenicity of nonhuman primate lentiviruses in their natural hosts might be due to low viral replication (Hartung et al., 1992). However, several subsequent studies as well as this study have shown that during the chronic phase of infection, primate lentiviruses replicate at moderate to high levels without clinical or pathological signs (Rey-Couille et al., 1998; Diop et al., 2000; Goldstein et al., 2000; Broussard et al., 2001; Holzammer et al., 2001). Therefore the lack of pathogenicity of SIVs, including SIVmnd-1, cannot be explained by limited replication in their natural host. Anti-gp36 and anti-V3 antibody titers were performed to evaluate antibody responses and relationship to virus load. The immunodominant region of the gp36 is one of the most conserved SIV epitopes and it is useful in diagnostic tests in both humans and nonhuman primates. Detecting anti-gp36 antibodies is a very effective diagnostic tool (Simon et al., 2001). Although in HIV-1 infection V3 region of gp120 is highly variable, this region is significantly more conserved in SIV infection (Franchini et al., 1990). This is supported
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by the finding that all the infected mandrills tested so far reacted with GB-1 V3 peptide. Our SIVmnd-1 V3 peptide is able to even detect antibodies in SIVmnd-2-infected mandrills or SIVdrl-infected drills (Souquiere et al., 2001; Simon et al., 2001). This is not surprising, as SIVmnd-2 and SIVdrl are recombinant viruses with a SIVmnd-1 envelope (Souquiere et al., 2001; Hu et al., 2003). The anti-V3 antibodies were tested because a decrease in titers is often correlated with disease progression and a decline in serum neutralizing activity in HIV-1-infected humans (Fenouillet et al., 1995). We have also reported a loss of anti-V3 antibodies in an SIVmnd-1-infected mandrill with immune suppression (Pandrea et al., 2001). However, none of the mandrills presented here showed decreased anti-V3 titers. Also, there was no clear correlation between viral load and anti-V3 titers. Theoretically, a more meaningful gauge of immune function and its relationship to viral load may have been neutralizing antibody responses. However, SIVmnd-1 GB1 grows poorly in cell culture, and this has hampered a proper evaluation of neutralizing antibody responses. Also, it should be noted that in other SIV naturally infected African monkeys, neutralizing antibody responses are generally low (Apetrei et al., 2004). From a clinical point of view, African monkeys may be similar to long-term nonprogressing HIV-infected humans. It was suggested that SIV adaptation to the nonprogressor status may be due to induction of a persistent infection with a symptom-free period exceeding the normal life span of most monkeys (Pandrea et al., 2001). However, in African nonhuman primates infected with SIV, the pathologic mechanisms must be different from those observed in HIV1-infected long-term nonprogressors, in whom the low HIV-1 loads are due to active cellular immune responses (Cao et al., 1995). In contrast, African monkeys have high viral loads which are associated with an attenuated antiviral cellular immune response (Chakrabarti et al., 2000a; Holznagel et al., 2002; Silvestri et al., 2003). Moreover, this equilibrium is maintained for long periods of time, without any clinical and immunological sign of immune suppression. Thus, our analysis of immunophenotypic markers in SIVmnd-1-infected mandrills was designed to determine if high levels of viremia have any effect on T cells. None of the parameters included in our immunophenotypic analysis showed significant differences between SIVmnd-1-infected and uninfected mandrills. Chronically infected mandrills were found to maintain relatively normal CD4⫹ counts in peripheral blood, despite years of infection. The slight increase in levels of CD8⫹ HLA-DR⫹ cells observed in chronically infected mandrills was not associated with decreased levels of CD8⫹ CD28⫹ cells, unlike results observed in humans (Giorgi et al., 1999; Sousa et al., 2002). However, this data are similar to data previously reported in SIVsm-infected sooty mangabeys (Silvestri et al., 2003). Our results indicate that SIVmnd-1 is well adapted to its mandrill host, and that cross-species transmission of lentiviruses in the wild can be controlled by the new primate
host. The mechanism(s) of this resistance to SIV of African monkeys should be further investigated to gain insights to control HIV infection.
Materials and methods Animals A total of 19 SIVmnd-1 chronically infected mandrills were examined, 4 of which resided in a large semi-free enclosure, and were naturally infected in captivity. Furthermore, these four (17B, 17D, 17D2, 17G) were offspring of the founder SIVmnd-1 GB1 female with whom they were raised/housed, suggesting that vertical transmission occurred. However, serial plasma samples were only examined on two of these mandrills: F17B (1992, 1993, 1995, 1999, and 2000) and F17D (1993, 1999, and 2000). Eleven mandrills were experimentally infected in two different experiments: mandrills 16E, 2C2, 12A4, 10G, 12C2 were intravenously infected with 3000 TCID50. Data on acute infection in these animals have been published elsewhere (Onanga et al., 2002). Mandrills 12A3, 16C, 12A7, 5I, 5J, 6B were infected intravenously with 300 TCID50 SIVmnd-1 and viral loads were measured (day 180). In both experiments, the inoculum was derived from plasma prepared as previously described (Onanga et al., 2002). An additional 11 uninfected mandrills were used as controls for flow cytometry analyses. In the study group, 5 animals were male (2C2, 12A4, 17G, 12C2, 10G) and 10 were female (17B, 17D, 17D2, 12A3, 16C, 16E, 12A7, 5I, 5J, 6B). The mean age of the animals was 8.6 (range 3–19). The mean weight for the males was 30 kg and 15 kg for females. Mandrill 12C2 died in 2000 of gastric dilatation, with no evidence of AIDS (Onanga et al., 2002). The wild mandrills originated from the Lope reserve in Central Gabon. All were adult solitary males in good health at the time of capture. No follow-up was possible for these mandrills because they were released. Sampling Animals were anesthetized with ketamine–HCl and 7–10 ml of whole blood was collected in EDTA. Plasma was separated and stored at ⫺80°C until used for semiquantitative viral load analysis and serology. Whole blood was used for flow cytometry as described below. Quantification of viral RNA in plasma RNA quantification was performed by a limiting-dilution RT-PCR assay specific for SIVmnd-1 (Onanaga et al., 2002). The primers were MP5 (5⬘-CCA GAT AAG TGG AAG ATA GAA AAG-3⬘) and MP6 (5⬘-GGG AGA TAT GGG AAG ATT GGG-3⬘), allowing the amplification of a 476-bp region within the SIVmnd-1 GB1 pol gene as pre-
I. Pandrea et al. / Virology 317 (2003) 119 –127
viously described (Nerrienet et al., 1998). Since the virus used in this study was derived from the SIVmnd-1 GB1, the possibility of mismatches was minimal. RNA was extracted from plasma using the QIAamp Viral RNA Mini kit (Qiagen, Courtaboeuf, France). The RNA extracted from 540 l of plasma was subjected to seven 5- to 10-fold serial dilutions which were used for the RT-PCR (one step RT-PCR kit, Qiagen). Reverse transcription was performed at 50°C for 30 min in the presence of MP5 and MP6, followed by 47 cycles of 95°C for 15 s, 55°C for 45 s, and 72°C for 1 min, with a final extension of 7 min at 72°C. The absolute target copy numbers were determined by amplification in parallel of known amounts of a SIVmnd-1 RNA external standard. The standard was obtained by amplifying the 476-bp region of SIVmnd-1 GB1 pol from F17 genomic DNA. The PCR product was cloned into the pCR 2.1 vector using the topoTA Cloning Kit (Invitrogen, Groningen, The Netherlands). The 5⬘-3⬘ sense orientation of the insert was verified by sequence analyses. A clone containing the insert in 5⬘-3⬘ orientation was chosen and subjected to PCR using the primer pair pCRT7S 5⬘ (GCG TAA TAC GAC TCA CTA TAG GGA GAG GA GCTC GAG CGG CCG CCA GTG TG) and pCRT7AS (5⬘-ATT ACG CCA AGC TTG GTA CCG AG3⬘), which hybridize to regions just upstream and downstream of the pCR2.1 plasmid polylinker sequence. Cycling conditions for this PCR consisted of an initial DNA denaturation step, followed by 40 cycles that included a denaturation step at 92°C for 30 s, primer annealing at 62°C for 45 s, and DNA synthesis at 72°C for 45 s, with a final extension of 7 min at 72°C. The resulting PCR product contained the cloned insert together with a T7 promoter sequence at its 5⬘ end. After clean up of the PCR product (PCR purification kit, Qiagen, Courtaboeuf, France) this DNA was used as a template for an in vitro transcription (MEGAscript kit, Ambion, Austin, TX). The resulting RNA was DNase digested and nonincorporated nucleotides were eliminated using a Chroma Spin-30 DEPC H20 Column (Clontech, Palo Alto, CA). Size and purity of the synthesized RNA were verified on a denaturating-PAGE gel. The concentration of the in vitro transcript was determined by a highly sensitive and specific Gene Quant II spectrophotometric operation system as already described (Onanga et al., 2002). Aliquots were stored at ⫺80°C until use. Immediately before usage, one aliquot of RNA was thawed and diluted in RNase free H2O and five dilutions of the RNA were used as external standard in each experiment. The sensitivity of the assay was 2 ⫻ 102 RNA copies/ml of plasma.
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Table 2 Antibody panel used for evaluating host immune dynamics in mandrills chronically infected with SIVmnd-1 mAB-PEa (clone, source) d
IgG1 (BD ) IgG1 (BD) IgG1 (BD) CD4 (Leu-3a BD) CD8 (Leu-2a BD) CD28 (Leu-28BD) CD28 (Leu-28BD) CD4 (Leu-3a BD) CD4 (Leu-3a BD) CD8 (Leu-2a BD)
mAB-FITCb (clone, source)
Cell type defined
IgG1 (PN0639 ITc) Control Control IgG1 (IT) Control IgG2a (BD) Control CD20 (Leu-16 BD) B cells CD3 (FN-18 BSe) T cells, T helper cells CD3 (FN-18 BS) T cells, T cytotoxic cells CD4 (Leu-3 BD) Costimulatory T helper cells CD8 (Leu 2a BD) Costimulatory T cytotoxic cells CD8 (Leu 2a BD) T helper, T cytotoxic cells HLA-DR (BD) Activated MHC II CD4⫹ cells HLA-DR (BD) Activated MHC II CD8⫹ cells
a
Phycoerythrin (PE). Fluorescein isothiocyanate (FITC). c ImmunoTech (IT). d BioSource (BS). e Becton Dickinson (BD). b
tested against gp36 and V3 peptide, respectively. The cutoff was established at 0.1 o.d. Flow cytometry analysis To quantify lymphocyte subsets and examine lymphocyte activation in infected versus uninfected animals, a variety of antibodies against cell-surface markers were used, as summarized in Table 2. Samples from noninfected (M5H, M2I, F12A3, F5J, F16C, F12A7, and F5I), and infected (F17, F17D, M2C2, M12A4, and M10G) mandrills were analyzed by flow cytometry. One hundred microliters of EDTA-treated whole blood was incubated in the dark with 5 l antibody for 30 min at 4°C. Red blood cells were lysed using a standard ammonium chloride lysing solution. Cells were then washed twice in phosphate-buffered saline (PBS) and resuspended in PBS with 1% paraformaldehyde. Data were acquired using a FACsCalibur flow cytometer (Immunocytometry System, Becton Dickinson) and analyzed with Cell Quest software (Immunocytometry System, Becton Dickinson). Statistical analysis and data presentation Flow cytometriy data were analyzed for significant differences (P ⬍ 0.05) using the Mann–Whitney test. Correlations were calculated and expressed as the Spearman coefficient of correlation (rs). Data were displayed using the box-and-whisker plot method (Fig. 1).
Primate immunodeficiency viruses ELISA (PIV-ELISA) Acknowledgments Antibody titers to both gp36 and V3 peptide were measured using a PIV-ELISA, as previous described (Simon et al., 2001). Twofold serial dilutions from 1/100 to 1/12,800 and from 1/100 to 1/43,200 were done for each sample
This work was supported by grants from Agence Nationale pour la Recherche sur le SIDA (ANRS) and NIH (RO1-AI44596).
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