While high dose mucosal challenge of SIV provides greater assurance that the SIV administration will result in successful infection, these doses exceed naturally occurring viral titers in body fluids [14
]. Due to the large number of virions in high dose inoculums, host defenses at mucosal sites may be overwhelmed, reducing our ability to detect the beneficial effects of vaccines or microbicides. Low dose viral administration more closely mimics naturally occurring viral inocula and, therefore, would be predicted to be more appropriate for vaccine, microbicide or transmission studies, although these experiments present additional issues both in designing and comparing studies (including inoculation timing, viral dose and optimal samples collection time points). In this study, the SIV-macaque model was utilized to evaluate viral and immune changes following either high or low dose SIV transmission via
the oral route. Virologic analyses revealed that plasma viral set-points during chronic phases were comparable between the low and high dosed macaques indicating that irrespective of the oral challenge dose the resultant viral replication is comparable during the chronic phase of the infection.
In general, the transcription changes of key immune modulators such as IFN-γ, TNF-α, IL-12 and IL-10 in PBMC following SIV infection in the low and high dosed orally SIVinfected macaques are in agreement with published studies [29
]. For example, the lack of an upregulation of the cytokines TNF-α and IL-10 during the chronic phase of the SIV infection in IV inoculated macaques has also been reported, although a transient upregulation of these cytokines can be observed in some macaques [70
]. Also, IFN-γ gene expression level in PBMCs is not upregulated following oral SIV-infected infant macaques [29
], in agreement with our findings. In lymph nodes, other studies have described an increase in IFN-γ, TNF-α, IL-12 and IL-10 gene expression within the first week post-SIV infection in orally or vaginally exposed macaques [29
]. However, the most dramatic upregulation was observed in lymph nodes near the sites of virus administration whereas those distal lymphoid tissues (similar to the ones assessed here) were less affected or unchanged [29
Assessment of immune modulators in PBMCs of the low and high dosed macaques identified two genes, OAS and CXCL10, that were upregulated. Overall, the mRNA levels of OAS and CXCL10 were similar in low and high dosed macaque PBMCs, although we did observe a more rapid increase in the high dosed (and intravenously inoculated) macaques in comparison to the low dosed macaques. These delayed immune responses occurred in low dosed macaques that had detectable plasma viral loads at the day 7 time point ( compared to ), and, therefore, can not be attributed solely to a delay in SIV replication. Indeed, a recent study by Liu et al
. assessed the impact of SIV dosage on viral and immunologic factors following rectal SIV infection of macaques [74
] and, although CXCL10 and OAS were not evaluated in that study, this group also found that low SIV dosages results in a delayed, as well as a reduced expression of a number of plasma cytokines and chemoki nes (IFN-α, IFN-γ, IL-1R α, IL-15, IL-18 and MCP-1) [74
]. Together, these data suggest a model in which low doses of SIV delivered mucosally result in a delayed innate immunologic response in peripheral blood when compared to macaques administered a high mucosal dose of SIV.
Assessment of peripheral lymph nodes following SIV infection in the low and high dosed macaques identified three immune modulators that were upregulated (OAS, CXCL9 and CXCL10). The immunologic differences between the differentially dosed macaques was most obvious within the lymph nodes in which high dosed macaques had increased OAS and CXCL10 expression while low dosed macaques upregulated CXCL9 and CXCL10 (, ). Interestingly, these two immune modulators are differentially regulated; OAS is produced in response to IFN-α stimulation [75
] and CXCL9 principally induced by IFN-γ [76
]. Interestingly, CXCL10 can be induced by both IFN-α and IFN-γ [76
]. Therefore, the differential immune modulator transcript expression may be driven by a strong IFN-α mediated response in high dosed macaques and a strong IFN-γ mediated response in low dosed macaques. Since OAS is an antiviral protein [75
] and the CXCL9 and CXCL10 are chemokines that bind the receptor CXCR3 [78
] expressed on Th1 (CD4+ and CD8+ T cells) as well as NK cells [80
], this differential expression has the potential to elicit different immune responses both within the peripheral lymph node as well as other lymphatic tissues. Our previous studies demonstrated that increased mRNA expression of OAS and CXCL10 at mucosal sites was associated with slower disease progression, whereas increased mRNA expression of these same immune factors in peripheral blood cells and lymph nodes was associated with more rapid disease progression [27
]. Therefore, the location of these immune modulators likely plays an important in determining early events following infection which has the potential to impact disease outcome.
Analysis of purified populations of CD3+ T cells and CD14+ monocytes determined that in PBMCs CD3+ T cells were the major producer of CXCL10 whereas OAS was produced mainly by CD14+ monocytes. Because CD4+ T cells can potentially be infected with SIV, it is possible that the increase in CXCL10 expression is due to direct viral infection, although indirect cytokine effects cannot be ruled out. In contrast, monocytes are rarely found to be infected with HIV. Accordingly, as monocytes produce OAS at higher levels than lymphocytes in response to interferon [82
], this suggests that interferon may be responsible for the monocyte-specific upregulation of OAS. These immunemodulators might be induced to defend against SIV infection, yet they could also potentially increase the activation state of CD4+ target cells, which would be predicted to facilitate HIV spread and increase the rate of disease progression.
In summary, the studies presented here are the first to provide a detailed assessment of immune and viral changes in Rhesus macaques inoculated orally with low and high doses of SIV. These studies provide evidence that infection after low dose SIV administration has more in common with natural HIV infection than does high dose SIV administration, particularly with regard to the limited number of virions establishing infection [45
]. Overall, the similarities in viral and immune parameters between the two SIV dosing strategies were more evident than the differences. The potential for an extended eclipse phase (delay in viral and innate immune responses) likely provides an additional opportunity for a vaccine to be protective when oral low dose SIV inoculations are administered. We also found differential patterns of gene expression observed in the lymph nodes of the high (upregulation of OAS and CXCL10) and low dosed (upregulation of CXCL9 and CXCL10) orally SIV inoculated macaques. These data would indicate that SIV challenge doses influence the innate immune response at least through the establishment of the chronic phase of the infection. Overall, these data support the use of low dose challenges in the SIV-macaque model as they establish virologic outcomes that are more comparable to what has been observed during mucosal HIV infections [45
] and therefore represents a better model for evaluating efficacy of vaccines and microbicides. Finally, these studies have the potential to inform future SIV/HIV vaccine efficacy trials in which vaccinated hosts have the potential to be infected with a range of viral challenge doses. The authors do not have any commercial or other considerations that might be interpreted as a conflict of interest with regard to the data presented herein.