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J Virol. 2010 May; 84(10): 5443–5447.
Published online 2010 March 10. doi:  10.1128/JVI.02452-09
PMCID: PMC2863841

High Viremia Is Associated with High Levels of In Vivo Major Histocompatibility Complex Class I Downregulation in Rhesus Macaques Infected with Simian Immunodeficiency Virus SIVmac239 [down-pointing small open triangle]


Human and simian immunodeficiency viruses (HIV and SIV) downregulate major histocompatibility complex class I (MHC-I) molecules from the surface of infected cells. Although this activity is conserved across viral isolates, its importance in AIDS pathogenesis is not clear. We therefore developed an assay to detect the level of MHC-I expression of SIV-infected cells directly ex vivo. Here we show that the extent of MHC-I downregulation is greatest in SIVmac239-infected macaques that never effectively control virus replication. Our results suggest that a high level of MHC-I downregulation is a hallmark of fast disease progression in SIV infection.

The level of HIV viremia in chronic infection is associated with the rate of progression to AIDS (13). In untreated HIV infection the median set point for viremia is ~30,000 viral RNA (vRNA) copy equivalents (Eq)/ml plasma, and the median time to AIDS is ~10 years (8, 12, 19). In contrast, some patients termed “rapid progressors” fail to resolve primary viremia and succumb to disease within 3 years, with virus loads of ≥100,000 copy Eq/ml (12). Rhesus macaques infected with simian immunodeficiency virus SIVmac239 show a similar range of outcomes. Most animals maintain chronic viremia between 0.1 and 5 million copy Eq/ml, mount detectable cellular immune responses to SIV antigens, and develop AIDS 1 to 2 years after infection. Like human rapid progressors, some animals have viremia >5 million copy Eq/ml, develop weak or undetectable cellular immune responses, and progress to AIDS within 6 months of infection.

How do viral and host factors combine to “set” these different set points? Nef is a multifunctional protein that is required for high-titer virus replication in vivo (6). One of its functions is to downregulate cell surface expression of major histocompatibility complex class I (MHC-I) antigens, an activity which is broadly conserved across immunodeficiency virus isolates (15, 18, 21). Nef-mediated downregulation of MHC-I protects infected cells from lysis by HIV-specific cytotoxic T lymphocytes (CTL) in in vitro assays (1, 4, 11, 22, 26). Although these data suggest that Nef-mediated immune evasion could play an important role in AIDS pathogenesis, there has been little direct evidence linking disease progression with MHC-I downregulation in vivo. HIV nef polymorphisms have been associated with different rates of disease progression (2, 10, 24), but it has been impossible to determine whether different levels of virus-mediated MHC-I downregulation for infected cells could contribute to rapid progression.

SIVmac239 nef mutants that abrogate MHC-I downregulating activity have been used in a few studies to infect macaques. In these animals, mutations restoring the ability to downregulate MHC-I occur shortly after infection, suggesting that this activity is favored by natural selection (14, 20). However, no study has been able to directly quantify MHC-I expression on virus-infected cells in vivo, and as a result the importance of MHC-I downregulation in AIDS pathogenesis has remained controversial. We hypothesized that unusually effective MHC-I downregulation on SIV-infected cells could interfere with the development of CTL responses, contributing to rapid progression in macaques. We therefore devised a novel assay to quantify MHC-I expression on SIV-infected cells directly ex vivo and used it to analyze the relationship among levels of cellular immune responses, viremia, and MHC-I downregulation in SIV-infected macaques.

Rapid progressors make weaker and more narrowly focused cellular immune responses to SIV than normal progressors.

We assembled a cohort of SIVmac239-infected Indian rhesus macaques whose chronic-phase virus loads ranged from 9.4 × 104 to 1.2 × 109 copy equivalents (Eq)/ml, including six animals with viremia close to or above 107 copy Eq/ml, which we termed rapid progressors (Fig. (Fig.1A).1A). Animals were assigned to protocols approved by the University of Wisconsin Institutional Animal Care and Use Committee and cared for according to the NIH Guide for the Care and Use of Laboratory Animals (14a). Data regarding SIVmac239 challenge, sample collection, and expressed MHC-I alleles (9) are summarized in Table S1 of the supplemental material. No animals in this study were vaccinated against SIV antigens.

FIG. 1.
SIVmac239 replication and frequencies of PBMC secreting IFN-γ in response to viral antigens in a cohort of typical and rapid progressor macaques. (A) Six of the 15 animals had viremia of >5 × 106 vRNA copy Eq/ml at 4 weeks postinfection ...

We assessed the magnitude and breadth of the animals' SIVmac239-specific immune responses by using a gamma interferon (IFN-γ) enzyme-linked immunospot assay as described elsewhere (25). Peptide pools used in these assays are detailed in Table S2 of the supplemental material. In early infection both rapid progressors and normal progressors had detectable immune responses. Rapid progressors recognized a median three distinct regions of the SIV proteome (range, zero to four) with a median total magnitude of 373 spot-forming cells (SFC)/million peripheral blood mononuclear cells (PBMC) (range, 0 to 1,035). Normal progressors made a median seven responses (range, 1 to 13) with a median total magnitude of 1,814 SFC/million PBMC (range, 265 to 2,925) (Fig. (Fig.1B).1B). In most normal progressors, both the breadth and magnitude of cellular immune responses increased during chronic infection (number of median responses, 8 [range, 1 to 26]; total magnitude of median responses, 2,810 SFC/million PBMC [range, 102 to 11,212]). In contrast, with one exception we could not detect SIV-specific cellular immune responses in chronically infected rapid progressors (Fig. 1B and C). Differences between rapid and normal progressors in the epitopic breadth and total magnitude of the SIV-specific cellular immune response were significant in both acute and chronic infections (Fig. 1B and C).

High postacute viremia is associated with preservation of wild-type nef sequence.

Nef is highly immunogenic, and in many animals Nef-specific CTL select for escape mutant viruses (16, 23). We reasoned that these escape mutations, while conferring the ability to avoid CTL detection, could also disrupt Nef functions, including MHC-I downmodulation. Therefore, we next asked whether particular nef substitutions observed in chronically infected animals in our cohort could be associated with the varied levels of plasma viremia. Virus isolated from animals in this cohort averaged 3.7 nonsynonymous substitutions in nef (Table (Table1),1), but no particular substitutions could be correlated with diminished MHC-I downregulation activity. However, virus from four of the six rapid progressors (rhAW61, r97009, r99030, and r98059) retained the wild-type SIVmac239 nef sequence. These were the only animals in the cohort whose virus harbored no mutations in nef.

Four of the six rhesus macaques with the highest viral load retained the wild-type nef sequence until their time of death

Ex vivo detection of SIVmac239-infected cells.

Since wild-type nef sequences were preserved in several rapid progressors, we hypothesized that virus in these animals might downregulate MHC-I to a greater extent than in normal progressors. To address this possibility, we devised an assay that allowed us to visualize MHC-I downregulation on SIV-infected cells ex vivo from lymph nodes. To enrich samples for infected cells, we depleted CD8+ T cells, NK cells, macrophages, and B cells by using magnetic beads (Miltenyi Biotec Inc., Auburn, CA). We stained the remaining cells for CD3 and CD4 and identified infected cells by using a monoclonal antibody directed against a conserved region of the Gag p27 protein (clone 55-2F12; AIDS Research and Reference Reagent Program) (Fig. (Fig.2A).2A). We used uninfected CD4+ T cells of the same sample as an internal control to quantify the level of MHC-I, CD3, and CD4 expression (Fig. (Fig.2B2B).

FIG. 2.
Flow cytometric detection of SIV-infected lymph node cells ex vivo and evaluation of cell surface marker downregulation. (A) Lymph node cells depleted of macrophages, B cells, NK cells, and CD8+ T cells were gated on lymphocytes by using forward ...

The majority of Gag p27+ cells had low CD3 expression (Fig. (Fig.2C)2C) and did not express CD4 (Fig. (Fig.2D),2D), in accord with previous studies showing that SIV downregulates these molecules on infected cells (17). In several animals Gag p27+ cells expressed measurably lower levels of MHC-I than did uninfected cells (Fig. (Fig.2e),2e), showing that our assay could detect MHC-I downregulation directly ex vivo.

The extent of MHC-I downregulation on SIV-infected cells is greatest in rapid progressors.

We next determined whether the level of MHC-I downregulation on infected cells differed between rapid progressors and normal progressors. To quantitate the extent of MHC-I downregulation we defined the relative expression of MHC-I on infected cells using the following equation: [(mean fluorescence intensity of MHC-I on p27+ cells)/(mean fluorescence intensity of MHC-I on p27 cells)] × 100.

We applied a similar approach to determine the relative expression of CD3 and CD4. The extent of MHC-I downregulation on SIV-infected cells varied among animals and in each animal was considerably less than the level of CD3 and CD4 downregulation. Nonetheless, the level of MHC-I downregulation on SIV-infected cells was significantly greater in the rapid progressor animals than in normal progressors (Fig. (Fig.3A).3A). In contrast, the extents of CD4 and CD3 downregulation on infected cells did not differ between groups (Fig. 3B and C).

FIG. 3.
Comparison of relative expression levels of various antigens on the surfaces of SIV-infected cells ex vivo. (A) MHC-I expression levels are significantly lower on infected cells from rapid progressor animals than on infected cells from normal progressors. ...

Our assay employed monoclonal antibody W6/32, the only clone available that binds macaque MHC-I molecules. It is important to note that W6/32 recognizes both classical (MHC-I A and B) and nonclassical (MHC-I E and I) proteins. As a result, we could not distinguish between MHC-I proteins sensitive to Nef-mediated downregulation (most Mamu-A and -B alleles) and those that resist downmodulation (Mamu-E and -I, as well as some -B alleles). Our assay is therefore likely not as sensitive as it would be if MHC-I A group-specific and/or B group-specific antibodies were available. Moreover, because Mamu-B alleles vary in their susceptibility to Nef-mediated downregulation (5), it is likely that animals' SIV-specific CTL repertoires are differentially impacted by this activity of Nef. We therefore refrain from using our results to derive a correlation between the extent of MHC-I downregulation and the breadth or magnitude of the SIV-specific CTL response. Nonetheless, our observations have afforded the first measurement of MHC-I expression levels on AIDS virus-infected cells directly ex vivo. They revealed that high levels of MHC-I downregulation on SIV-infected cells are associated with uncontrolled virus replication and a lack of strong SIV-specific immune responses. Our results therefore raise the possibility that expression of MHC-I molecules that are particularly sensitive to MHC-I downregulation may play a role in rapidly progressive AIDS virus infection.

Supplementary Material

[Supplemental material]


This work was supported by NCRR grant P51 RR000167 and was conducted at a facility constructed with support from grants RR15459 and RR020141.

We are grateful to David I. Watkins for support and helpful discussions.


[down-pointing small open triangle]Published ahead of print on 10 March 2010.

Supplemental material for this article may be found at


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