Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Genetic Analyses of HIV-1 env Sequences Demonstrate Limited Compartmentalization in Breast Milk and Suggest Viral Replication within the Breast That Increases with Mastitis

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na⁺) concentration. The association between milk Na⁺ and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na⁺ was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.Breastfeeding accounts for 30 to 50% of mother-to-child-transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) (38). MTCT through breastfeeding occurs primarily in sub-Saharan Africa, where the use of artificial infant formula is often not feasible because of cost and the associated infant mortality from infections due to the use of unsafe water and the lack of the protective effects of breast milk (19, 38, 51). Numerous strategies to reduce postnatal HIV-1 infection of infants while preserving the advantages of breastfeeding have been evaluated, including maternal use of combination antiretroviral therapy or infant antiretroviral prophylaxis during the period of breastfeeding (5, 25, 26, 30, 40). Understanding the biologic events that increase the concentration of HIV-1 in breast milk is critical to the development and evaluation of interventions to reduce postnatal MTCT.The risk of MTCT is strongly associated with the concentration of HIV-1 in breast milk (28, 46, 47). Although breast milk HIV-1 RNA concentrations correlate with those in plasma, levels in milk are typically 2 log₁₀ lower (15, 24, 43). This suggests that HIV-1 in blood and milk may not mix freely, likely because of the closure of tight junctions between mammary alveolar cells that occurs once milk production is established and before weaning (16). Thus, HIV-1 may evolve in the breast without substantial mixing with blood, i.e., evolving viral variants would become compartmentalized—a phenomenon that has been observed in the central nervous system (50) and in some studies of the genital tract (10, 44, 57). Compartmentalization of HIV-1 variants has been detected in the breast milk of a small number of women (3, 4), but other data suggest that compartmentalization in breast milk may be uncommon (22).Breast inflammation (mastitis) occurs frequently during lactation, most commonly without symptoms. Mastitis is associated with elevations in HIV-1 RNA levels in milk (15, 31, 47, 55), an increase in the number of inflammatory cells in milk, and opening of tight junctions in the mammary epithelium that allows passage of subcellular blood components, of which sodium (Na⁺) serves as a marker (15, 16, 36, 47, 55). Greater permeability of mammary epithelia may allow the passage of free virus from the blood into breast milk, which would result in the mixing of HIV-1 subpopulations from blood and milk. Alternatively, inflammation in the breast may induce replication of virus by HIV-1-infected cells within the breast, which would result in divergence between milk and blood HIV-1 subpopulations. Here we describe detailed genetic analyses of HIV-1 subpopulations in the blood and breast milk to determine whether mastitis affects the structure of these populations and to gain understanding of the processes that may lead to increased concentrations of HIV-1 in milk.     

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.HIV-1-specific cytotoxic T lymphocytes (CTLs) have an important role in the control of HIV-1 replication during acute and chronic phases of an HIV-1 infection (5, 28, 33). On the other hand, HIV-1 can escape from the host immune system by various mechanisms. These may include the appearance of HIV-1 carrying escape mutations in its immunodominant CTL epitopes as well as Nef-mediated downregulation of HLA class I molecules. There is a growing body of evidence for the former mechanism, i.e., that CTLs targeting immunodominant HIV-1 epitopes select escape mutants in chronically HIV-1-infected individuals (18, 20, 36), whereas the latter mechanism was proved by demonstrating that HIV-1-specific CTLs fail to kill Nef-positive-HIV-1-infected CD4⁺ T cells but effectively kill Nef-defective-HIV-1-infected ones or that they suppress the replication of Nef-defective HIV-1 much more than that of Nef-positive HIV-1 (12, 13, 42, 45).It is speculated that HIV-1 immunodominant epitope-specific CTLs have the ability to suppress HIV-1 replication and effectively select escape mutants. However, the correlation between this ability of the CTLs and the appearance of escape mutants is still unclear, because it is not easy to evaluate the ability of HIV-1-specific CTLs to exert a strong immune pressure in vivo. To examine this ability, most previous studies measured the number of HIV-1-specific CTLs or CD8⁺ T cells and the CTL activity against target cells prepulsed with the epitope peptide or those infected with HIV-1 recombinant vaccinia virus (6, 7, 23, 46). However, the results obtained from such experiments do not reflect the ability of the CTLs to exert immune pressure in vivo. We and other groups previously utilized an assay to directly evaluate the ability of the CTLs to suppress HIV-1 replication in vitro (1, 17, 18, 42, 43). This assay may be better for evaluation of immune pressure by HIV-1-specific CTLs than other assays, because the ability of the CTLs to suppress HIV-1 replication is directly measured in cultures of HIV-1-infected CD4⁺ T cells incubated with HIV-1-specific CTL clones. But it still remains unknown whether this assay reflects immune pressure in vivo.In the present study, we investigated whether HIV-1-specific CTLs having a strong ability to suppress HIV-1 replication could positively select escape mutants. Since HLA-A*1101 is known to be an HLA allele relatively associated with a slow progression to AIDS (32), it is speculated that some HLA-A*1101-restricted CTLs would have a strong ability to suppress HIV-1 replication in vitro. Therefore, we first focused on 4 well-known HLA-A*1101-restricted CTL epitopes in the present study. We investigated the frequency of CTLs specific for these epitopes in chronically HIV-1-infected individuals, the ability of these CTLs to suppress HIV-1 replication in vitro, and whether the escape mutants were selected by the CTLs. Furthermore, we analyzed the expression of Programmed Death-1 (PD-1) on these CTLs ex vivo and antigen recognition of them.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

Protective HLA Class I Alleles That Restrict Acute-Phase CD8+ T-Cell Responses Are Associated with Viral Escape Mutations Located in Highly Conserved Regions of Human Immunodeficiency Virus Type 1

The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles suggests that some CD8⁺ T-cell responses may be more effective than others at containing HIV-1. Unfortunately, substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to identify responses most critical to this control. It has been proposed that CD8 responses targeting conserved regions of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations in these regions may significantly impair viral replication. To address this hypothesis at the population level, we derived near-full-length viral genomes from 98 chronically infected individuals and identified a total of 76 HLA class I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution. The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated mutations in the absence of the selecting HLA allele was also commonly observed, suggesting an impact of most CTL escape mutations on viral replication. Although no correlations were observed between the number or location of HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase immunodominant responses revealed a strong positive correlation between mutations at conserved residues and protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of these regions in the design of an effective vaccine.Despite substantial advances in antiretroviral therapies, development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine remains a critical goal (6, 39, 82). Unfortunately, current vaccine efforts have failed to reduce infection rates in humans (9, 75) and have only achieved modest decreases in viral loads in the simian immunodeficiency virus (SIV)/SHIV macaque model (21, 44, 81). A majority of these vaccine approaches have focused on inducing T-cell responses, utilizing large regions of the virus in an attempt to induce a broad array of immune responses (6, 34, 44, 81). While it is well established that CD8⁺ T-cell responses play a critical role in the containment of HIV-1 (45, 49, 67), supported in part by the strong association of particular HLA class I alleles with control of HIV (20, 33, 42, 61), it remains unclear which particular CD8⁺ T-cell responses are best able to control the virus and thus should be preferentially targeted by a vaccine. Studies comparing the magnitude, breadth, and function of CD8⁺ T-cell responses in subjects exhibiting either enhanced or poor control of HIV-1 have yielded few clues as to the specific factors associated with an effective CD8⁺ T-cell response (2, 28, 64, 67). Various differences in the functional capacity of T-cell responses have been observed in long-term nonprogressors (1, 26, 64), although it is possible that these differences may be reflective of an intact immune response, as opposed to having had directly enhanced immune control. As such, efforts are needed to identify factors or phenotypes associated with protective CD8⁺ T-cell responses in order to enable vaccines to induce the most effective responses.Recent studies have begun to suggest that the specificity of the CD8⁺ T-cell response, or the targeting of specific regions of the virus, may be associated with control of HIV-1. Preferential targeting of Gag, a structurally conserved viral protein responsible for multiple functions, has been associated with lower viral loads (25, 43, 56, 60, 77, 85). Furthermore, Kiepiela et al. (43) recently illustrated in a large cohort of 578 clade C-infected subjects that Gag-specific responses were associated with lowered viremia, in contrast to Env-specific responses, which were associated with higher viremia. These data are in line with previous observations that many of the major histocompatibility complex (MHC) class I alleles most strongly associated with control of HIV-1 and SIV, namely, HLA-B57, HLA-B27, and Mamu-A*01, restrict immunodominant CD8⁺ T-cell responses against the Gag protein (8, 10, 24, 63, 68, 83). However, other alleles associated with slower disease progression, such as HLA-B51 in humans and Mamu-B08 and B-17 in the rhesus macaque, do not immunodominantly target Gag, suggesting that targeting of some other regions of the virus may also be capable of eliciting control (8, 52-54). In addition, recent studies investigating the pattern of HIV-1-specific CD8⁺ T-cell responses during acute infection reveal that only a small subset of CD8⁺ T-cell responses restricted by any given HLA allele arise during acute infection and that there exist clear immunodominance patterns to these responses (8, 77, 85). Since control of HIV-1 is likely to be established or lost during the first few weeks of infection, these data suggest that potentially only a few key CD8⁺ T-cell responses may be needed to adequately establish early control of HIV-1.One of the major factors limiting the effectiveness of CD8⁺ T-cell responses is the propensity for HIV-1 to evade these responses through sequence evolution or viral escape (3, 13, 66). Even single point mutations within a targeted CD8 epitope can effectively abrogate recognition by either the HLA allele or the T-cell receptor. However, recent studies have begun to highlight that many sequence polymorphisms will revert to more common consensus residues upon transmission of HIV-1 to a new host, including many cytotoxic T-lymphocyte (CTL) escape mutations (4, 30, 33, 48, 50). Notably, the more rapidly reverting mutations have been observed to preferentially occur at conserved residues, indicating that structurally conserved regions of the virus may be particularly refractory to sequence changes (50). In support of these data, many CTL escape mutations have now been observed to directly impair viral replication (15, 23, 55, 74), in particular those known to either revert or require the presence of secondary compensatory mutations (15, 23, 73, 74). Taken together, these data suggest that, whereas CTL escape mutations provide a benefit to the virus to enable the evasion of host immune pressures, some of these mutations may come at a substantial cost to viral replication. These data may also imply that the association between Gag-specific responses and control of HIV-1 may be due to the targeting of highly conserved regions of the virus that are difficult to evade through sequence evolution.The propensity by which HIV-1 escapes CD8⁺ T-cell responses, and the reproducibility by which mutations arise at precise residues in targeted CD8 epitopes (3, 48), also enables the utilization of sequence data to predict which responses may be most capable of exerting immune selection pressure on the virus. Studies in HIV-1, SIV, and hepatitis C virus (16, 58, 65, 78) are now rapidly identifying immune-driven CTL escape mutations across these highly variable pathogens at the population level by correlating sequence polymorphisms in these viruses with the expression of particular HLA alleles. We provide here an analysis of HLA-associated mutations across the entire HIV-1 genome using a set of sequences derived from clade B chronically infected individuals. Through full-length viral genome coverage, these data provide an unbiased analysis of the location of these mutations and suggest that the control of HIV-1 by particular HLA alleles correlates with their ability to preferentially restrict early CD8⁺ T-cell responses capable of selecting for viral escape mutations at highly conserved residues of the virus. These data provide support for the inclusion of specific highly conserved regions of HIV-1 into vaccine antigens.     

Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4⁺ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.Human immunodeficiency virus type 1 can disseminate between and within hosts by cell-free infection or by direct cell-cell spread. Cell-cell spread of HIV-1 between CD4⁺ T cells is an efficient means of viral dissemination (65) and has been estimated to be several orders of magnitude more rapid than cell-free virus infection (6, 8, 41, 64, 74). Cell-cell transmission of HIV-1 takes place at the virological synapse (VS), a multimolecular structure that forms at the interface between an HIV-1-infected T cell and an uninfected target T cell during intercellular contact (27). Related structures that facilitate cell-cell spread of HIV-1 between dendritic cells and T cells (42) and between macrophages and T cells (16, 17) and for cell-cell spread of the related retrovirus human T-cell leukemia virus type 1 (HTLV-1) (24) have also been described. Moreover, more long-range cell-cell transfer can occur via cellular projections, including filopodia (71) and membrane nanotubes (75). The VS is initiated by binding of the HIV-1 envelope glycoprotein (Env), which is expressed on the surfaces of infected T cells, to HIV-1 entry receptors (CD4 and either CXCR4 or CCR5) present on the target cell membrane (6, 22, 27, 41, 61, 73). Interactions between LFA-1 and ICAM-1 and ICAM-3 further stabilize the conjugate interface and, together with Env receptor binding, help trigger the recruitment of viral proteins, CD4/coreceptor, and integrins to the contact site (27, 28, 61). The enrichment of viral and cellular proteins at the VS is an active process, dependent on cytoskeletal remodeling, and in the infected T cell both the actin and tubulin network regulate polarization of HIV-1 proteins at the cell-cell interface, thus directing HIV-1 assembly and egress toward the engaged target cell (27, 29). Virus is transferred by budding into the synaptic cleft, and virions subsequently attach to the target cell membrane to mediate entry, either by fusion at the plasma membrane or possibly following endocytic uptake (2, 22). In this way, the VS promotes more rapid infection kinetics and may enhance HIV-1 pathogenesis in vivo.Cells have evolved a number of barriers to resist invading microorganisms. One mechanism that appears to be particularly important in counteracting HIV-1 infection is a group of interferon-inducible, innate restriction factors that includes TRIM5α, APOBEC3G, and tetherin (38, 49, 69, 79). Tetherin (BST-2/CD317) is a host protein expressed by many cell types, including CD4⁺ T cells, that acts at a late stage of the HIV-1 life cycle to trap (or “tether”) mature virions at the plasma membranes of virus-producing cells, thereby inhibiting cell-free virus release (49, 56, 81). This antiviral activity of tetherin is not restricted to HIV-1, and tetherin can also inhibit the release of other enveloped viruses from infected cells (31, 40, 54, 62). What the cellular function of tetherin is besides its antiviral activity is unclear, but because expression is upregulated following alpha/beta interferon (IFN-α/β) treatment (1) and tetherin can restrict a range of enveloped viruses, tetherin has been postulated to be a broad-acting mediator of the innate immune defense against enveloped viruses.To circumvent restriction of particle release, HIV-1 encodes the 16-kDa accessory protein Vpu, which antagonizes tetherin and restores normal virus budding (47, 78). The molecular mechanisms by which Vpu does this are not entirely clear, but evidence suggests that Vpu may exert its antagonistic function by downregulating tetherin from the cell surface, trapping it in the trans-Golgi network (10) and targeting it for degradation by the proteasome (12, 39, 81) or lysosome (9, 25, 44); however, degradation of tetherin may be dispensable for Vpu activity (13), and in HIV-1-infected T cells, surface downregulation of tetherin has been reported to be minor (45), suggesting that global removal of tetherin from the plasma membrane may not be necessary to antagonize its function.Tetherin-mediated restriction of HIV-1 and antagonism by Vpu have been the focus of much research, and inhibition of cell-free virus infection has been well documented (33, 47-49, 77, 81, 82). In contrast, less studied is the impact of tetherin on direct cell-cell dissemination. For example, it is not clear if tetherin-mediated restriction inhibits T cell-T cell spread as efficiently as cell-free release or whether tetherin affects VS formation. To address these questions, we analyzed Vpu⁺ and Vpu⁻ viruses for their ability to spread directly between Jurkat T cells and primary CD4⁺ T cells in the presence or absence of endogenous tetherin. Our data suggest that tetherin does not restrict HIV-1 in the context of cell-to-cell transmission of virus between T cells expressing endogenous tetherin. Interestingly, we also that observed that Vpu-defective virus may disseminate more efficiently by cell-cell spread at the VS. We postulate that cell-cell spread may favor viral pathogenesis by allowing HIV-1 to disseminate in the presence of tetherin during an interferon-producing innate response.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.There is broad heterogeneity in the ability of HIV-infected individuals to control virus replication, ranging from elite controllers, who maintain undetectable viral loads without treatment, to rapid progressors, who progress to AIDS within 2 years of infection (9, 22, 32). Many interrelated factors, including host and viral genetic factors involved in antiviral immunity and the viral life cycle, may partially account for the differences in the course of disease progression (10, 11, 30, 41). The complex interplay between host genetic factors and viral factors is exemplified by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocyte (CTL) responses, which exert considerable immune pressure on the virus, resulting in escape mutations that affect the interaction of viral and host proteins, thereby influencing infection outcome.The exact mechanisms by which some HLA class I alleles, such as HLA-B*57 and HLA-B*27, are associated with slower progression to AIDS, while others, such as B*5802 and B*18, are associated with accelerated disease progression (6, 20, 42), are unclear. The magnitude and/or breadth of HLA-restricted CTL responses to the conserved Gag protein has been correlated inversely with disease progression or markers of disease progression in several studies (12, 21, 28, 31, 35, 43, 46), although there are some exceptions (4, 16, 37), while preferential targeting of the highly variable envelope protein (as occurs in HLA-B*5802-positive individuals) correlates with higher viral loads (21, 29). Protective HLA alleles restrict CTL responses that impose a strong selection pressure on a few specific Gag p24 epitopes, resulting in escape mutations (14) for which fitness costs have been demonstrated either through site-directed mutations introduced into a reference strain background (2, 8, 25, 38) or through in vivo reversion of these mutations after transmission to an HLA-mismatched individual (8, 24). Recent evidence suggests that Gag escape mutations with a fitness cost, particularly those in p24, are a significant determinant of disease progression: the transmitted number of HLA-B-associated polymorphisms in Gag was found to significantly impact the viral set point in recipients (although an associated fitness cost was not shown) (7, 15), and in a small number of infants, decreased fitness of the transmitted virus with HLA-B*5703/5801-selected mutations in Gag p24 epitopes resulted in slower disease progression (33, 39). Also, the number of reverting Gag mutations (thought to revert as a consequence of fitness costs) associated with individual HLA-B alleles was strongly correlated with the HLA-linked viral set point in chronically infected patients (26). A recent in vitro study showed that HLA-associated variation in Gag-protease, with resulting reduced replication capacity, may contribute to viral control in HIV-1 subtype B-infected elite controllers (27). Taken together, these studies suggest that CTL responses restricted by favorable HLA alleles select for escape mutations in conserved epitopes, particularly those in Gag, resulting in a fitness cost to HIV and therefore at least partly explaining the slower disease progression in individuals carrying these alleles.To date, many of the studies investigating the fitness cost of Gag escape mutations and their clinical relevance have concentrated on escape mutations associated with protective HLA alleles, have not assessed fitness consequences in the natural sequence background (in the presence of other escape and compensatory mutations), and/or have focused on a limited number of patients. Most importantly, the majority of studies have focused on HIV-1 subtype B. The present study is the first to use a large population-based approach and clinically derived Gag-protease sequences to investigate comprehensively the relationships between immune-driven sequence variation in Gag, viral replication capacity, and markers of disease progression in chronic infection with HIV-1 subtype C, the most predominant subtype in the epidemic. We assayed the replication capacity of recombinant viruses encoding patient Gag-protease in an HIV-1-inducible green fluorescent protein (GFP) reporter cell line and found associations between lower replication capacities, protective HLA alleles, protective HLA-associated mutations, lower baseline viral loads, and higher baseline CD4 counts. However, Gag-protease replication capacity did not correlate with the subsequent rate of CD4 decline.