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Because most studies of AIDS pathogenesis in nonhuman primates have been performed in Indian-origin rhesus macaques (Macaca mulatta), little is known about lentiviral pathogenicity and control of virus replication following infection of alternative macaque species. Here, we report the consequences of simian-human immunodeficiency virus SHIV-89.6P and SIVmac251 infection in cynomolgus (Macaca fascicularis) and rhesus macaques of Chinese origin. Compared to the pathogenicity of the same viruses in Indian rhesus macaques, both cynomolgus and Chinese rhesus macaques showed lower levels of plasma virus. By 9 to 10 months after infection, both viruses became undetectable in plasma more frequently in cynomolgus than in either Chinese or Indian rhesus macaques. Furthermore, after SHIV-89.6P infection, CD4+ T-cell numbers declined less and survival was longer in cynomolgus and Chinese rhesus macaques than in Indian rhesus macaques. This attenuated pathogenicity was associated with gamma interferon ELISPOT responses to Gag and Env that were generated earlier and of higher frequency in cynomolgus than in Indian rhesus macaques. Cynomolgus macaques also developed higher titer neutralizing antibodies against SHIV-89.6 at 10 and 20 weeks postinoculation than Indian rhesus macaques. These studies demonstrate that the pathogenicity of nonhuman primate lentiviruses varies markedly based on the species or geographic origin of the macaques infected and suggest that the cellular immune responses may contribute to the control of pathogenicity in cynomolgus macaques. While cynomolgus and Chinese rhesus macaques provide alternative animal models of lentiviral infection, the lower levels of viremia in cynomolgus macaques limit the usefulness of infection of this species for vaccine trials that utilize viral load as an experimental endpoint.
Nonhuman primates infected with naturally occurring or genetically engineered lentiviruses serve as important animal models for evaluating the efficacy of candidate AIDS vaccines (9, 14). Studies in which macaques are immunized with new vaccine prototypes and subsequently challenged with pathogenic lentiviruses help to determine which vaccine products are best advanced to human clinical trials. The vast majority of AIDS vaccine/challenge data has been generated in rhesus macaques of Indian origin. An extensive knowledge of the genetics of Indian rhesus macaques has allowed detailed characterizations of major histocompatibility complex (MHC)-restricted immune responses elicited during vaccination and challenge (1, 17). However, infection of alternative macaque species such as cynomolgus or rhesus macaques from different geographic origins could provide information on comparative pathogenicity and provide an opportunity to examine diverse responses to viral infections.
Rhesus macaques of Chinese origin, both wild and captive, are more plentiful than Indian origin rhesus macaques. Preliminary observations suggest that important genetic differences may exist between regional populations of rhesus macaques that could affect the biological consequences of viral infections (8, 20). Indeed, others have demonstrated that rhesus macaques of Chinese origin have lower plasma virus levels and a more attenuated pathogenicity than rhesus macaques of Indian origin when infected with some primate lentiviruses (10, 11, 19). Other macaque species, such as cynomolgus macaques, are more readily available than rhesus macaques and can be infected with the lentiviruses routinely employed in AIDS vaccine challenge studies. However, little is known about the relative pathogenicity and the immune responses to the well-characterized experimental lentiviruses in this species. Identifying relevant and readily available nonhuman primate models of lentivirus infection will help provide information needed for developing an effective AIDS vaccine.
In the present study, we assessed pathogenicity and virus-specific immunity induced by the acutely pathogenic chimeric simian-human immunodeficiency virus SHIV-89.6P and the pathogenic SIVmac251 in Chinese-origin rhesus and cynomolgus macaques, comparing these findings with those seen in Indian-origin rhesus macaques. Compared to infection of Indian-origin rhesus macaques, infection of Chinese-origin rhesus macaques or cynomolgus macaques with either virus resulted in attenuated pathogenicity, as evidenced by lower levels of viremia and/or less profound CD4+ T lymphopenia, better preservation of virus-specific immune responses, and prolonged survival. Interestingly, the attenuated pathogenicity of these viruses in cynomolgus macaques was associated with early and strong virus-specific cellular immune responses. Although the lower levels of plasma viremia could limit the usefulness of other macaque species in vaccine challenge studies that utilized viral load as an experimental endpoint, they may also provide alternative models of lentiviral infection that display viral loads more similar to those seen in human infection. Further, understanding the immune correlates of this attenuation may aid in designing effective strategies for immune prophylaxis.
SHIV-89.6P was derived as previously described by serial passage of SHIV-89.6 in Indian-origin rhesus macaques (16). The viral stock was prepared by ex vivo propagation of blood, lymph node, and splenic lymphocytes obtained during the final in vivo passage. SIVmac251 was derived from an Indian-origin rhesus macaque that developed a B-cell lymphoma (2). The SIV stocks used to inoculate animals had been passaged four or five times in human peripheral blood mononuclear cells (PBMC).
Naïve Chinese-origin rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) from Mauritius were used in this study. Comparative data from Indian-origin rhesus macaques were obtained from the control groups of vaccine and other pathogenesis studies in which animals received a control treatment or no treatment at the time of virus inoculation. The origin of rhesus macaques from either the Indian or Chinese subcontinent of Asia was confirmed by animal import permits, purchase records, or pedigrees. For the Chinese rhesus and cynomolgus groups, plasma viral load, CD4 T-cell count, ELISPOT, and neutralizing antibodies were measured in all animals. For the Indian rhesus macaques comparator groups, ELISPOT and neutralizing antibody titers were available for only a subset of animals. All animals were inoculated intravenously with approximately 100 50% monkey infectious doses of SIVmac251 or SHIV-896P. The same SHIV-89.6P stock was used for all animals. All Chinese rhesus macaques and cynomolgus macaques and 6/19 Indian rhesus macaques were inoculated with the same SIVmac251 stock; 13/19 Indian rhesus macaques were inoculated with an earlier or later passage of the same SIV stock. All animals were observed for 270 to 300 days postinoculation or until moribund.
Animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School or Bioqual, Inc., and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academic Press, Washington, DC, 1996).
Plasma levels of SHIV-89.6P or SIVmac 251 RNA were measured using the branched DNA amplification assay (Bayer Diagnostics, Berkeley, CA) that detects gag sequences shared by the two viruses.
CD4 T-cell percentages were derived by flow cytometric analysis of blood lymphocytes using a whole blood lysis technique and anti-CD3-allophycocyanin (FN18 or SP34) and anti-CD4-phycoerythrin (19Thy5D7 or L200). CD4 T-cell percentages were converted to absolute counts using the absolute lymphocyte number derived using an automated hematology instrument (T540, Beckman Coulter, Miami, Florida, or ADVIA 120, Bayer Diagnostics, Tarrytown, New York).
Antibody-mediated neutralization of SHIV-89.6P or SIVmac 251 was assessed in CEMx174 (for SIV) or MT-2 (for SHIV) cell killing assay as previously described (13). Briefly, 50 μl of cell-free virus containing 500 50% tissue culture infectious doses (TCID50s) were added to multiple dilutions of test plasma in 100 μl of growth medium (RPMI 1640/12% fetal bovine serum/50 μg gentamicin) in triplicate in 96-well culture plates. The mixtures were incubated for 1 h at 37°C followed by the addition of CEMx174 or MT-2 cells (5 × 104 in 100 ul) to each well. Infection led to extensive syncytium formation and virus-induced cell killing in approximately 4 to 6 days in the absence of antibodies.
Neutralization was measured by staining viable cells with Finter's neutral red in poly-l-lysine-coated plates. Percent protection was determined by calculating the difference in absorption (A540) between test wells (cells plus serum plus virus) and virus control wells (cells plus virus), dividing this result by the difference in absorption between cell control wells (cells only) and virus control wells, and multiplying by 100. Neutralizing titers are give as the reciprocal dilution required to protect 50% of cells from virus-induced cell killing. Cell-free stock of SIVmac was prepared in H9 cells and SHIV-89.6P was prepared in human PBMC.
ELISPOT assays were performed by stimulating unfractionated PBMC with SIV Gag peptide pools and either SIVmac251 Env pools or HIV-89.6 Env pools. Ninety-six-well Multiscreen HA plates (Millipore, Bedford, Massachusetts) were coated overnight (100 μl/well) at 4°C with mouse anti-human gamma interferon IFN-γ monoclonal antibody (B27; BD PharMingen, San Diego, California) at 10 μg/ml in endotoxin-free Dulbecco's phosphate-buffered saline (D-PBS, Life Technologies, Gaithersburg, Maryland). Plates were washed three times with D-PBS containing 0.25% Tween-20 (D-PBS/Tween), blocked for 2 h at 37°C with 100 μl/well D-PBS containing 5% fetal bovine serum, and rinsed with RPMI containing 10% fetal bovine serum to remove Tween 20.
PBMC were plated in triplicate at 2 × 105/well in 100-μl final volume with either medium alone or 2 μg/ml SIVmac239 Gag, SIVmac239 Env, or HIV-1 89.6 Env peptide pools. The peptide pools consisted of overlapping 15-mer peptides spanning the SIVmac239 Gag, Env, or HIV-1 89.6 Env proteins, and were used such that each peptide was present at a concentration of 2 μg/ml. Following 18 h incubation at 37°C, the plates were washed nine times with D-PBS/Tween and once with distilled water. The plates were then incubated with 2 μg/ml biotinylated rabbit anti-human IFN-γ antibody (Biosource, Camarillo, California) for 2 h at room temperature, washed six times with Coulter Wash (Beckman Coulter, Miami, Florida), and incubated for 2 h with a 1:500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology, Birmingham, Alabama). Following five washes with Coulter Wash and one with D-PBS, the plates were developed with nitro blue tetrazolium/5-bromo-4-chloro-3 indolylphosphate chromogen (Pierce, Rockford, Illinois), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Hitech Instruments, Edgement, Pennsylvania). The mean number of spots from triplicate wells was calculated for each animal after subtracting the mean number of spots from the medium-alone control wells and adjusted to represent the mean number of spots per 106 PBMC.
Plasma virus levels were transformed to log10 copies/ml before all analyses. Plasma virus and CD4 T-cell count data were reduced to six measures for each animal. Baseline CD4 T-cell number was measured between days 0 and 2 postinoculation. If more than one measurement was available, the median was used. Peak viral load was the maximum value measured between days 7 and 17 inclusive. For postacute CD4 T cells count and viral load measures, we calculated the median of all values between days 35 and 77 inclusive. For long-term set point CD4 T-cell count and viral load, we calculated the median of all values between days 84 and 300 inclusive. To assess neutralizing antibody and ELISPOT responses, specimens from single time points corresponding to the same three phases of infection described above (specifically, 2 to 4 weeks, 8 to 10 weeks, and 15 to 20 weeks postinoculation) were analyzed. The Kruskall-Wallis test was used to assess overall differences between the three species for each CD4 T-cell count, viral load, neutralizing antibody titer and ELISPOT measure. If this result was significant, then two post hoc analyses using the Wilcoxon rank sum test were performed with a Bonferroni adjustment to allow for multiple comparisons. Spearman's rank correlation was used to assess the association between variables. Survival was plotted using the method of Kaplan and Meier, and significance between groups was assessed using the log rank test. Statistical significance was defined as P < 0.05.
SHIV-89.6P and SIVmac251 are challenge viruses commonly employed in nonhuman primate vaccine studies, and infection of Indian rhesus macaques with these viruses has been well characterized. To determine how these viruses replicate in cynomolgus and Chinese rhesus macaques, we measured plasma virus and CD4+ T-cell number in these alternative macaque models, comparing these values to historical data from Indian-origin rhesus macaques infected with the same viruses.
To compare plasma virus levels, we determined three measures of viral load: (i) the peak level achieved during primary infection (typically achieved at days 10 to 17), (ii) the level of plasma virus during the postacute period (median of days 35 to 77 postinoculation) and (iii) the long-term set point level (median of days 84 to 300). A smoothed average of plasma virus level for each group is illustrated for SHIV-89.6P and SIVmac251 infection (Fig. (Fig.1A1A and and1B).1B). These three measures of plasma virus levels observed after inoculation of cynomolgus and Chinese-origin rhesus macaques were compared with those observed in Indian-origin rhesus macaques (Tables (Tables11 and and2).2). Median plasma virus levels of SHIV-89.6P were significantly lower in cynomolgus than in Indian rhesus macaques in all three postinoculation time periods and in Chinese rhesus macaques after peak. The same trend was observed after inoculation with SIVmac251, although plasma virus levels in Chinese rhesus macaques and cynomolgus monkeys were significantly lower only during the postacute period.
During the 280- to 300-day observation period following virus inoculation, the level of plasma viremia fell and remained below detection (<500 RNA copies/ml for at least two consecutive measurements) in 75% (6/8) of cynomolgus macaques infected with SHIV-89.6P, whereas plasma virus became undetectable in 12.5% or less of the Chinese (1/8) or Indian rhesus macaques (2/10) (Fig. (Fig.2).2). SIVmac251 viremia became undetectable in 50% (4/8) of cynomolgus but remained above >500 copies/ml in all Chinese-origin (0/8) and Indian-origin (0/19) rhesus macaques.
We performed a similar comparison of absolute CD4+ T-cell number in cynomolgus, Chinese-origin rhesus macaques and Indian-origin rhesus macaques during the same postacute and long-term set point periods. Smoothed averages of CD4+ T-cell counts for each group after inoculation with SHIV-89.6P or SIVmac251 are illustrated in Fig. Fig.1A1A and and1B,1B, respectively. Preinoculation baseline levels of CD4+ T cells did not differ between the three groups, although Indian rhesus macaques tended toward higher CD4+ T-cell counts than the other two groups. Infection with the acutely cytopathic SHIV-89.6P precipitously reduced CD4+ T cells in all animals. However, the CD4+ T-cell levels in cynomolgus and Chinese rhesus macaques during the postacute and long-term set point periods were significantly higher than those observed in Indian rhesus macaques infected with SHIV-89.6P (Table (Table1).1). Postinoculation levels of CD4+ T cells in all monkeys infected with SIVmac251 were lower than their preinoculation levels, and the same trend in magnitude of CD4 T lymphopenia was seen. However, the magnitude of the CD4+ T-cell decline was not nearly as dramatic as that seen following SHIV-89.6P infection, and neither Chinese rhesus macaques nor cynomolgus macaques differed significantly from Indian rhesus macaques (Table (Table2).2). These results document attenuated pathogenicity of both viruses in cynomolgus and Chinese rhesus macaques as measured by reduced SIV and SHIV replication and modulation of SHIV-induced CD4+ T lymphopenia.
We next compared the immune response generated in rhesus macaques and cynomolgus macaques after infection with these viruses by measuring both humoral and cellular immune responses to the inoculating viruses. Neutralizing antibody titers against the infecting viruses were measured at three selected time points after inoculation (Fig. (Fig.3).3). Titers against SIVmac251 were measured using a T-cell line-adapted stock of this virus which is more sensitive to neutralization than SIV primary isolates (12).
Using this assay, neutralizing titers against SIVmac251 were generated by 4 weeks in most animals irrespective of species (Fig. (Fig.3B).3B). There was a trend for anti-SIV titers to increase over the 16-week study period in Indian rhesus macaques and for titers to decrease in cynomolgus macaques over the same period. These changes were likely a result of higher levels of SIV replication in Indian rhesus macaques compared to cynomolgus macaques. Neutralizing antibody titers against SHIV-89.6P were not measurable in any animal until 8 to 10 weeks postinoculation (Fig. (Fig.3A).3A). At 15 to 16 weeks postinoculation, anti-SHIV titers were significantly higher in both Chinese rhesus macaques and cynomolgus than in Indian rhesus macaques. Of the 20 Indian-origin rhesus macaques infected with SHIV-89.6P, 16 failed to generate measurable neutralizing antibodies, whereas one of eight Chinese rhesus macaques and zero of eight cynomolgus macaques failed to develop neutralizing antibody responses. The failure of most Indian rhesus macaques to generate neutralizing antibodies probably occurred as a result of the more profound CD4 T lymphopenia that occurred in rhesus macaques derived from this geographic location; those Indian rhesus macaques that did generate neutralizing antibodies had the best preservation of CD4+ T cells.
Cellular immune responses to these viruses were quantified by ELISPOT assays in which unfractionated PBMC were stimulated in vitro to produce IFN-γ using Gag and Pol peptide pools that were homologous with the infecting virus. Cynomolgus macaques infected with either SHIV-89.6P or SIVmac251 generated strong ELISPOT responses against viral peptides by 2 weeks postinoculation (Fig. (Fig.4A4A and and4B);4B); these responses were significantly higher than those observed in Indian rhesus macaques. The ELISPOT responses then decreased somewhat in cynomolgus at the later time points. In contrast, ELISPOT responses in Chinese rhesus macaques infected with SHIV-89.6P or SIVmac251 increased progressively over the study period. At week 20, median responses in SHIV-infected Chinese rhesus macaques were significantly higher than those in Indian rhesus macaques. ELISPOT responses remained low in Indian rhesus macaques infected with SHIV-89.6P at all time points. Indian rhesus macaques infected with SIVmac251 developed ELISPOT responses by week 10 that were not significantly different from those seen in Chinese rhesus macaques or cynomolgus macaques. The early and strong cellular immune responses observed in cynomolgus macaques after infection with either virus may have contributed to the attenuating the pathogenicity of these viruses.
The early and strong cellular immune responses observed in cynomolgus macaques after infection with either virus were unique to this species. In the SHIV-89.6P-infected cohorts, these virus-specific cellular immune responses preceded neutralizing antibodies by several weeks. As another means to assess whether these early cellular immune responses in cynomolgus macaques may have contributed to more efficient control of lentivirus replication and attenuated of pathogenicity in this species, we tested whether a correlation existed between gamma interferon ELISPOT number and plasma virus at the 2-week postinoculation time point.
ELISPOT responses failed to correlate with plasma SHIV or SIV level in the Indian and Chinese rhesus macaque groups (Table (Table3,3, Fig. Fig.5).5). However, a trend toward a negative correlation between cellular immune response and viral load (although not statistically significant) was evident in cynomolgus macaques infected with either SHIV-89.6P or SIVmac251 (P = 0.07 and 0.06, respectively). These results emphasize the potential importance of early cellular immune responses in the ultimate clinical outcome of lentivirus infection and suggest that important species-related differences may exist in the kinetics of these responses.
We compared the survival of Chinese rhesus macaques and cynomolgus macaques with Indian rhesus macaques for 270 to 300 days after infection with either SIVmac251 or SHIV-89.6P. Survival was significantly shorter in Indian rhesus macaques inoculated with SHIV-89.6P than in either cynomolgus or Chinese rhesus macaques inoculated with the same virus (Fig. (Fig.6A,6A, P = 0.002). The same trend was seen in monkeys inoculated with SIVmac251, although differences in survival were not statistically significant (Fig. (Fig.6B,6B, P = 0.20). These results were consistent with the differences in viral replication and CD4+ T-cell loss between viruses and between the different macaque groups.
In an effort to identify other sources of macaques that may serve as useful animal models for AIDS vaccine studies, we characterized the course of infection of SIVmac251 and SHIV-89.6P in cynomolgus and Chinese rhesus macaques. When compared to Indian rhesus macaques, the pathogenicity of both viruses was moderately attenuated in Chinese rhesus macaques and markedly attenuated in cynomolgus macaques. Manifestations of attenuated pathogenicity included lower levels of plasma viremia, preservation of CD4+ T-cell number, preservation of both cellular and humoral immune responses, and increased survival time. We note, however, that cynomolgus macaques used in our study were of Mauritian origin. The pathogenicity of these viruses may differ in cynomolgus macaques from different Asian locations. However, the consequences of lentiviral infection we report in Chinese rhesus macaques and Mauritian cynomolgus macaques are in agreement with those reported in other studies of Chinese rhesus macaques and cynomolgus macaques that evaluated different viruses or employed other measures of pathogenicity (7, 10, 11, 19).
No existing nonhuman primate AIDS model is ideal for testing the efficacy of candidate HIV vaccines. Marked antigenic differences exist between the envelope glycoproteins of the SIVs and HIV-1. Infection of Indian-origin rhesus macaques with most SIVs also results in levels of plasma virus well above those seen in HIV-1 infection of humans. Alternatively, chimeric SHIVs share envelope composition with HIV-1. Infection with some SHIVs also results in an acute CD4 T lymphocytopenia that can serve as an additional experimental endpoint not available in SIV models. However, for some SHIVs such as SHIV-89.6P, the coreceptor usage and acute pathogenicity also differs from that seen in natural HIV-1 infection. Thus, infection of macaques with these chimeric viruses has also been criticized as a suboptimal model for AIDS vaccine testing (4).
Our results and those previously reported by others clearly indicate that the alternate nonhuman primate models such as cynomolgus or Chinese rhesus macaques macaque infected with SIV develop plasma virus levels and an attenuated disease course that approximates those seen in HIV infection (10). However, it is crucial that the nonhuman primate models used to evaluate vaccine efficacy provide experimental endpoints, such as plasma virus level or changes in CD4+ T-cell number, that have sufficient power to detect differences in vaccine effect while utilizing reasonable group sizes. It is readily apparent, based on the present data, that vaccine challenge studies utilizing cynomolgus macaques are not feasible since a large fraction of animals naturally control SIV or SHIV replication to levels that are below conventional detection; such studies would need to be very large to have reasonable power to detect a treatment-induced effect. Chinese rhesus macaques may be somewhat more useful in this regard. Although levels of plasma virus were lower than those seen in Indian rhesus macaques, SIV and SHIV viremia remained detectable in most animals.
Of particular interest in this study was the rapid and strong gamma interferon ELISPOT responses observed after both SHIV and SIV inoculation of cynomolgus macaques. These early virus-specific cellular immune occurred well before the generation of neutralizing antibodies, consistent with the hypothesis that attenuation of pathogenicity was due to immune control of virus replication mediated by T cells. If this relationship is causal, the present observation provides further evidence for the importance of the control of primary viremia as a determining factor in disease course.
Alternatively, SIV and SHIV may have inherently low replication rates in the lymphocytes and macrophages of cynomolgus macaques due to virus-host interactions unrelated to the immune responses induced by viral infection. Thus, viral dynamics in cynomolgus macaques may have resulted in reduced virally induced cytopathicity giving rise to better preservation of immune responses and, ultimately, an attenuation of disease course compared to that seen in Indian rhesus macaques. Indeed, there is evidence that pathogenicity of other viral, bacterial, and parasitic microbes is also attenuated in cynomolgus macaques (5, 6, 18).
However, three observations support a role for early T-cell immune responses in the attenuation of viral pathogenicity in cynomolgus macaques. First, attenuation of disease was observed after infection with both SIV and SHIV—viruses that infect different CD4+ T-cell subsets (15). Second, the early and strong ELISPOT responses also occurred in cynomolgus macaques infected with either SIV or SHIV. Finally, the trend toward a negative correlation between the magnitude of the cellular immune response and level of viremia 2 weeks postinoculation seen in cynomolgus macaques infected with either virus suggests that the reduction in virus replication may have resulted from the early cellular immune response.
Both SIVmac251 and SHIV-89.6P viruses used in these studies were either isolated from or propagated by serial in vivo passage in rhesus macaques of Indian origin (2, 16). Propagation or serial passage of viral stocks in Indian rhesus macaques may have resulted in the selection of viral variants adapted for more efficient replication and/or enhanced pathogenicity these animals compared to other cynomolgus or Chinese rhesus macaques. Indeed, not all chimeric SHIVs show such a large variation in pathogenicity when inoculated into different macaque species as we observed in this study (3). Therefore, serial in vivo passage of these two viruses in cynomolgus or Chinese rhesus macaques might result in virus isolates that replicate to higher levels and are more pathogenic in macaques of this species or geographic origin.
While these studies do not definitively elucidate the mechanism responsible for SIV and SHIV attenuation in cynomolgus and Chinese rhesus macaques, they do provide further evidence that therapeutic interventions or vaccination aimed at limiting lentivirus replication during primary infection have the potential to modulate disease progression later in the course of infection. Understanding the limitations of each nonhuman primate AIDS model is essential for the rational and efficient design of vaccine challenge studies.
This work was supported in part by NIH grants HL059747 (K.A.R.), NS037654 (K.C.W.), NS040237 (K.C.W.), and RR000168, by Harvard Medical School Center for AIDS Research (CFAR) grant AI060354, and by NCI/SAIC Frederick contract no. 23XS046A.