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.     

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.     

The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

The Na⁺-dependent K⁺ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K⁺-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K⁺ uptake and in modulating Na⁺ dependency. Expression of KtrB alone in a K⁺ uptake-deficient Escherichia coli strain conferred low K⁺ uptake activity that was not stimulated by Na⁺. Coexpression of both KtrA and KtrE with KtrB increased the K⁺ transport activity in a Na⁺-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K_m for K⁺ in all cases. However, none of the mutations eliminated the Na⁺ dependency of Ktr-mediated K⁺ transport. Mutations of residues on the cytoplasmic side had larger effects on K⁺ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K⁺ uptake rate and conferred Na⁺ dependency.Cyanobacterium Synechocystis sp. strain PCC 6803 contains a number of different K⁺ uptake systems that may contribute to satisfying its requirement of K⁺ (3, 19, 36). Among these systems, Ktr has been shown to have a major role not only in K⁺ uptake but also in adaptation against high-osmolarity stress (3, 19). Inactivation of the ktr gene renders the cells hypersensitive to high concentrations of NaCl and the nonionic compound sorbitol. Ktr-mediated K⁺ uptake depends on the presence of Na⁺ in the medium, which is likely to be an adaptation to salinity stress. A requirement of Na⁺ for K⁺ transport activity has also been found in the homologous protein from Vibrio alginolyticus (21). This dependency on Na⁺ is a unique property of Ktr-type transporters and has not been found in other types of K⁺ transporters or channels (32). The structure and function of Ktr-type transporters have been studied in a number of organisms (3, 6, 7, 9, 11-14, 18-20, 30, 32-34). The Ktr system from Synechocystis consists of three subunits, KtrA, KtrB, and KtrE (19). The KtrE gene and the KtrB gene form a cistron, whereas the KtrA gene resides at a site distant from the KtrEB genes in the Synechocystis genome (19). KtrB, the K⁺-translocating subunit, is a member of the Ktr/Trk/HKT family of K⁺ transporters. These transporters have been proposed to have evolved from two membrane-spanning K⁺ channels (6, 7). According to the model, this type of transporter contains eight transmembrane domains, which consist of a 4-fold-repeated membrane-pore-membrane (M1-P-M2) motif (6, 7, 13, 18). An intramolecular electrostatic interaction of Synechocystis KtrB has been proposed to stabilize the protein in its active configuration (12). In addition, a conserved His in the external region in Synechocystis KtrB has been shown to be crucial for KtrB function (39). The region of the Vibrio Ktr protein responsible for gating of ion permeation has been identified (9). However, not much is known about the mechanism of Na⁺ binding to KtrB in Synechocystis.The KtrA subunit belongs to the family of KTR (K⁺-transport nucleotide binding)/RCK (regulating the conductance of K⁺ channels) proteins, which contain a Rossmann-fold sequence encoding β-α protein structure for NAD⁺/NADH binding (17). Accordingly KtrA has been proposed to regulate the K⁺ transport activity of KtrB by changing its binding from NAD⁺ to NADH through a ligand-mediated conformational switch mechanism (25). It has also been shown that ATP promotes complex formation between KtrA and KtrB and that KtrAB from V. alginolyticus when expressed in Escherichia coli cells requires both ATP and the membrane potential for its activity (17).KtrE is a unique subunit found only in Synechocystis; it is not involved in KtrB-mediated K⁺ transport in V. alginolyticus and Bacillus subtilis (11, 32). The termination codon of ktrE overlaps the initiation codon of ktrB in the same cistron, which has not been found in other bacterial ktrB-related genes. Coexpression of KtrA with KtrB alone does not complement the growth defect of an E. coli K⁺ uptake mutant. However, introduction of KtrE into the same mutant background in addition to KtrA and KtrB complements the mutation of the K⁺ uptake system (19). Interestingly, the KtrE protein has been shown to function as a digalactosyldiacylglycerol (DGDG) synthase (EC 2.4.6.241), an enzyme that produces DGDG from monogalactosyldiacylglycerol (MGDG). KtrE has therefore also been designated DgdA (1). Under nonstress conditions, DGDG is found in the thylakoid membranes, which helps stabilize the photosystem II complex in Synechocystis (29). Under phosphate-limited conditions, DGDG is synthesized instead of phospholipids in Synechocystis (1). However, KtrB functions as a major K⁺-conducting transport pore in the Synechocystis plasma membrane. The subcellular localization of KtrE has not been identified directly. Inactivation of ktrE (also called dgdA) in Synechocystis does not result in sensitivity to osmotic stress imposed by 300 mM sorbitol (1). This may be inconsistent with the requirement of KtrE for KtrB-mediated K⁺ uptake in the presence of KtrA in the E. coli expression system (19).Because of these uncertainties about the roles of the KtrA and KtrE subunits in K⁺ uptake by KtrB in Synechocystis and about the identity of the Na⁺ binding site in KtrB, we examined the subcellular localization and membrane association of KtrA and KtrE, the requirement of these subunits for KtrB-mediated K⁺ uptake, and the primary target for Na⁺ binding in KtrB.     

Effective Simian Immunodeficiency Virus-Specific CD8+ T Cells Lack an Easily Detectable,Shared Characteristic

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8⁺ T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8⁺ T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8⁺ T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8⁺ T cells to suppress viral replication from SIVmac239-infected CD4⁺ T cells. Using this assay, we established an antiviral hierarchy when we compared CD8⁺ T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8⁺ T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8⁺ T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8⁺ T-cell response may be comprised of several factors.CD8⁺ T cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.     

Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection

The kinetics of CD8⁺ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8⁺ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4⁺ T cells early after SIV infection.The antiviral activity of AIDS virus-specific CD8⁺ T cells is well documented in both in vivo (1, 4, 21) and in vitro (8, 24, 29) studies. Accordingly, human immunodeficiency virus (HIV) vaccine modalities that focus on engendering antiviral CD8⁺ T cells are being developed (13, 26, 28). Ideally, a CD8⁺ T cell-based vaccine would stimulate responses against epitopes that are presented by major histocompatibility complex class I (MHC-I) molecules early after infection of a target cell. However, successful selection of antigenic sequences for a CD8⁺ T cell-based vaccine has been frustrated in part by an incomplete understanding of the properties of effective CD8⁺ T cell responses (25).     

Defective Human Immunodeficiency Virus-Specific CD8+ T-Cell Polyfunctionality,Proliferation, and Cytotoxicity Are Not Restored by Antiretroviral Therapy

Identifying the functions of human immunodeficiency virus (HIV)-specific CD8⁺ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8⁺ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8⁺ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4⁺ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8⁺ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8⁺ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8⁺ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8⁺ T-cell compartment that persist under conditions of low levels of antigen.Understanding the features of an effective immune response to human immunodeficiency virus (HIV) is among the most important goals for the design of HIV vaccines and immunotherapies. Most HIV-infected patients develop persistent viremia and CD4⁺ T-cell decline in the absence of antiviral therapy. However, evidence that immunologic control of HIV is possible can be drawn from a small group of rare patients who maintain normal CD4⁺ T-cell counts and restrict HIV replication to below 50 copies/ml plasma for up to 25 years without antiretroviral therapy (ART) (4, 22, 31, 40). Historically, these unique individuals were included within heterogeneous cohorts referred to as long-term survivors or long-term nonprogressors (LTNP), categorized solely based on their disease-free survival exceeding 7 to 10 years and their stable CD4⁺ T-cell counts (21). Over time, it became apparent that only a small subset of individuals within these cohorts had truly nonprogressive infection, maintaining good health with nondeclining CD4⁺ T-cell counts, and these true nonprogressors tended to have HIV type 1 (HIV-1) RNA levels below the lower detection limits of the newly available assays (23, 31). Some investigators have adopted other designations more recently, including elite controllers, elite suppressors, or HIV controllers. These designations vary by institution and, in some cases, rely only upon viral load measurements without a requirement for stable CD4⁺ T-cell counts (4, 22, 40). However, for our designation of true LTNP, we employ the inclusion criteria of stable health, nondeclining CD4⁺ T-cell counts, and maintenance of plasma viral RNA levels below 50 copies/ml without ART (29-31).Several lines of evidence strongly suggest that CD8⁺ T cells mediate this control of HIV in LTNP. HLA B*5701 is highly overrepresented in these patients, and in B*5701⁺ patients, the HIV-specific CD8⁺ T-cell response is largely focused on peptides restricted by the B57 protein (15, 31). In addition, similar control of simian immunodeficiency virus replication has been described in rhesus macaques carrying the Mamu B*08 or B*17 allele (25, 49). In these macaques, CD8⁺ T-cell depletion studies have strongly suggested that control of viral replication is mediated by CD8⁺ T cells (14). Although these results support the idea that CD8⁺ T cells are responsible for immunologic control, the mechanism remains incompletely understood.Several lines of evidence suggest that immunologic control in LTNP is not simply due to differences in autologous virus recognition by CD8⁺ T cells. The frequencies of CD8⁺ T cells specific for HIV or individual HIV-encoded gene products in the peripheral blood are not different in LTNP and untreated progressors (reviewed in reference 32). Putative “escape” mutations are found in viruses of both HLAB*57⁺ LTNP and HLA-matched progressors (4, 6, 28, 33, 34). In addition, comparable frequencies of CD8⁺ T cells of LTNP and progressors recognize autologous CD4⁺ T cells infected with the autologous virus (12, 28). Similar observations have recently been made in the rhesus macaque model (26). Collectively, these observations strongly suggest that features of the CD8⁺ T-cell response associated with immunologic control are not due to quantitative differences in the numbers of HIV-specific cells or to differential abilities of the autologous virus gene products to be recognized between patient groups.Several qualitative features in the HIV-specific CD8⁺ T-cell response have been associated with immunologic control in LTNP. LTNP have been found to have higher frequencies of “polyfunctional” CD8⁺ T cells, named for their ability to degranulate and produce multiple cytokines, including interleukin-2 (IL-2) (2, 5, 51). However, these cells comprise an extremely small proportion of the HIV-specific CD8⁺ T-cell response. In addition, there is considerable overlap between patient groups, and many LTNP have few or no such cells. Compared to those of progressors, HIV-specific CD8⁺ T cells of LTNP have a dramatically higher proliferative capacity, a greater ability to upregulate granzyme B (GrB) and perforin production, and a greater cytolytic capacity against autologous HIV-infected CD4⁺ T cells (3, 17, 24, 29, 30). Increased HIV-specific CD8⁺ T-cell proliferative capacity in LTNP compared to progressors has also been associated with lower PD-1 expression or IL-2 production by HIV-specific CD4⁺ or CD8⁺ T cells (11, 24, 48, 51).Considerable controversy exists over the cause-and-effect relationships between these qualitative differences in the CD8⁺ T-cell response and HIV viremia between patient groups. High levels of antigen can have potent effects on diverse cell types in humans and in animal models. For HIV, lowering the level of viremia through ART has been observed to increase the function of CD4⁺ and CD8⁺ T cells, NK cells, monocytes, and plasmacytoid dendritic cells (16, 18, 20, 37, 41, 45-47, 50). However, the vast majority of treated progressors will not control HIV replication when ART is interrupted (7, 9, 35), suggesting that many of the qualitative differences in the CD4⁺ or CD8⁺ T-cell response between LTNP and untreated progressors are not the cause of control over HIV but rather are likely an effect of viremia. In some but not all studies, ART was sufficient to restore the proliferative capacity, phenotype, and cytokine production by CD4⁺ T cells to levels similar to responses to other viruses or to the HIV-specific response of LTNP (13, 16, 18, 20, 37, 46, 50). Because better IL-2 production or function of HIV-specific CD4⁺ T cells has been associated with increased CD8⁺ T-cell proliferative capacity (24), it has also been suggested that diminished proliferative capacity of progressor CD8⁺ T cells may be an effect of viremia during the chronic phase of infection. In some studies, ART is sufficient to increase the frequency of polyfunctional HIV-specific CD8⁺ T cells or to decrease PD-1 expression (30, 41). However, the interpretations of the observations within these studies have relied on extrapolations between studies based upon cohorts with differing levels and durations of viral suppression or on examination of a limited number of functions or subsets in either CD4⁺ or CD8⁺ T cells.In the present study, we extended our earlier work and comprehensively examined a broad array of functions of HIV-specific T cells derived from two large patient groups, LTNP and progressors on ART, who possess comparable levels of HIV viremia as determined by a sensitive single-copy assay. In response to autologous HIV-infected CD4⁺ T cells, HIV-specific CD8⁺ T-cell proliferative capacity, IL-2 responsiveness, surface phenotype, PD-1 expression, polyfunctionality, and cytotoxic capacity were measured in considerable detail. We observe that although ART results in restoration of many of these functions, HIV-specific CD8⁺ T-cell polyfunctionality and proliferative and killing capacities are not restored to levels observed in LTNP.     

Impact of HLA in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

A broad Gag-specific CD8⁺ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8⁺ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8⁺ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fisher''s exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8⁺ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.Human immunodeficiency virus (HIV)-specific CD8⁺ T cells play a central role in controlling viral replication (12). It is the specificity of the CD8⁺ T-cell response, particularly the response to Gag, that is associated with low viral loads in HIV infection (7, 17, 34). Although immune control is undermined by the selection of viral mutations that prevent recognition by the CD8⁺ T cells, evasion of Gag-specific responses mediated by protective class I HLA-B alleles typically brings a reduction in viral replicative capacity, facilitating subsequent immune control of HIV (2, 20, 21). The same principle has been demonstrated in studies of simian immunodeficiency virus infection (18, 22).Recent studies showed that the class I HLA-B alleles that protect against disease progression present more Gag-specific CD8⁺ T-cell epitopes and drive the selection of more Gag-specific escape mutations than those alleles that are associated with high viral loads (23). These protective HLA-B alleles not only are beneficial to infected individuals expressing those alleles but also benefit a recipient following transmission, since the transmitted virus carrying multiple Gag escape mutations may have substantially reduced fitness (3, 4, 8). However, there is no benefit to the recipient if he or she shares the same protective allele as the donor because the transmitted virus carries escape mutations in the Gag epitopes that would otherwise be expected to mediate successful immune control in the recipient (8, 11).The sharing of HLA alleles between donor and recipient occurs frequently in mother-to-child transmission (MTCT). The risk of MTCT is related to viral load in the mother, and a high viral load is associated with nonprotective alleles, such as HLA-B*18 and -B*5802. This may contribute in two distinct ways to the more rapid progression observed in pediatric HIV infection (24, 26, 27). First, because infected children share 50% or more of their HLA alleles with the transmitting mother, they are less likely than adults to carry protective HLA alleles (16). Thus, infected children as a group carry fewer protective HLA alleles and more nonprotective HLA alleles. Second, even when the child has a protective allele, such as HLA-B*27, this allele does not offer protection if the maternally transmitted virus carries escape mutations within the key Gag epitopes that are presented by the protective allele (11, 19).However, it is clear that infected children who possess protective alleles, such as HLA-B*27 or HLA-B*57, can achieve durable immune control of HIV infection if the virus transmitted from the mother is not preadapted to those alleles (6, 10). HIV-specific CD8⁺ T-cell responses are detectable from birth in infected infants (32). Furthermore, as in adult infection (3, 8), HIV-infected children have the potential to benefit from transmission of low-fitness viruses in the situation where the mother possesses protective HLA alleles and the child does not share those protective alleles. MTCT of low-fitness viruses carrying CD8⁺ T-cell escape mutations was recently documented (28; J. Prado et al., unpublished data).In this study, undertaken in Durban, South Africa, we set out to test the hypothesis that HIV-infected children are less likely to progress rapidly to disease if either the infected child or the transmitting mother possesses a protective HLA allele that is not shared. The HLA alleles most strongly associated with low viral loads and high CD4 counts in a cohort of >1,200 HIV-infected adults in Durban are HLA-B*57 (-B*5702 and -B*5703), HLA-B*5801, and HLA-B*8101 (16; A. Leslie et al., unpublished data). These four alleles all present Gag-specific CD8⁺ T-cell epitopes, and in each case the escape mutations selected in these epitopes reduce viral replicative capacity (2-4, 8, 21, 23).Analyzing a previously described cohort of 61 HIV-infected children in Durban (24, 26, 32), South Africa, who were all monitored from birth, we first addressed the question of whether possession of any of these four alleles by either mother or child is associated with slower disease progression in the child and then determined whether sharing of protective alleles by mother and child affects the ability of the child to make the Gag-specific CD8⁺ T-cell responses restricted by the shared allele.     

Infection with “Escaped” Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08⁺) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8⁺ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8⁺ T-cell epitopes to investigate the contribution of epitope-specific CD8⁺ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08⁺ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08⁺ macaques. The five most immunodominant Mamu-B*08-restricted CD8⁺ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08⁺ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08⁺ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8⁺ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08⁺ macaques.A few individuals spontaneously control the replication of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) to very low levels. The precise mechanisms underlying this control are of great interest, as a clear understanding of what constitutes a successful immune response may aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which proteins to use as immunogens, the extent to which increasing the breadth and magnitude of responses is advantageous, how immunodomination affects T-cell responses, and if biasing the immune response toward particular effector profiles is beneficial. Characterization of immune responses made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation and evaluation.HIV ECs are generally not infected with grossly unfit viruses (6, 42). Instead, elite control of immunodeficiency virus replication is correlated with the presence of particular major histocompatibility complex class I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with the control of viremia implicates CD8⁺ T cells as being mediators of this immune containment. Several lines of evidence support this hypothesis. These lines of evidence include the correlation between the appearance of CD8⁺ T-cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that alleles associated with viral control restrict dominant acute-phase CD8⁺ T-cell responses (3), and the finding that responses directed against epitopes restricted by these alleles frequently select for viral escape variants (4, 27, 38). Perhaps most compelling is the observation that for a few HIV-infected individuals, the selection of escape variants by an immunodominant HLA-B27-restricted T-cell response temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51), recent data have found normal, high levels of replication in vivo upon the transmission of some of these variants (15).The association of control with MHC-I alleles does not, of course, implicate solely CD8⁺ T cells. MHC-I molecules are also ligands for killer immunoglobulin receptors (KIRs), which are predominantly expressed on natural killer (NK) cells. Genetic studies of HIV-infected humans suggest a model in which individuals with particular KIR/HLA combinations are predisposed to control HIV replication more readily than those with other KIR/HLA combinations (36, 37). These data were supported by functional studies of this KIR/HLA pairing in vitro, which demonstrated an inhibition of HIV replication by such NK cells (2). The relative contributions of NK and CD8⁺ T-cell responses to control have yet to be elucidated and may be closely intertwined.Previously, the experimental depletion of circulating CD8⁺ cells from SIVmac239-infected ECs resulted in a sharp spike in viremia, which resolved as CD8⁺ cells repopulated the periphery (19). During the reestablishment of control of SIV replication, CD8⁺ T cells targeting multiple epitopes restricted by alleles associated with elite control expanded in frequency, providing strong circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8 depletion antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication (19). It was therefore difficult to make definitive conclusions regarding the separate contributions of these subsets to maintaining the control of SIV replication in vivo.Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the macaque MHC-I allele most tightly associated with the control of SIVmac239, Mamu-B*08. Approximately 50% of Mamu-B*08-positive (Mamu-B*08⁺) animals infected with SIVmac239 become ECs (32). Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27. Although these two MHC-I genes are dissimilar in domains that are important for peptide binding, each molecule can bind peptides that are presented by the other molecule (33). This striking similarity suggests that the elite control of SIVmac239 in Mamu-B*08⁺ animals is a good model for the elite control of HIV.Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08⁺ rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by 2 amino acids in the α1 and α2 domains [9, 32]), escape has been documented only for SIV-infected Mamu-B*03⁺ macaques (16). Variation in these CD8⁺ T-cell epitopes accumulates with different kinetics, starting during acute infection for those targeted by high-magnitude responses.In this study, we addressed the question of whether the elite control of SIVmac239 in Mamu-B*08⁺ animals is mediated by the known high-frequency CD8⁺ T-cell responses targeting Mamu-B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the goal of reducing or abrogating immune responses directed against these epitopes during acute infection. We hypothesized that Mamu-B*08⁺ macaques would be unable to control SIV replication without these Mamu-B*08-restricted T-cell responses.     

Impact of Cytotoxic-T-Lymphocyte Memory Induction without Virus-Specific CD4+ T-Cell Help on Control of a Simian Immunodeficiency Virus Challenge in Rhesus Macaques

Despite many efforts to develop AIDS vaccines eliciting virus-specific T-cell responses, whether induction of these memory T cells by vaccination before human immunodeficiency virus (HIV) exposure can actually contribute to effective T-cell responses postinfection remains unclear. In particular, induction of HIV-specific memory CD4⁺ T cells may increase the target cell pool for HIV infection because the virus preferentially infects HIV-specific CD4⁺ T cells. However, virus-specific CD4⁺ helper T-cell responses are thought to be important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction in HIV infection, and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination without HIV-specific CD4⁺ T-cell help can exert effective responses after virus exposure. Here we show the impact of CD8⁺ T-cell memory induction without virus-specific CD4⁺ T-cell help on the control of a simian immunodeficiency virus (SIV) challenge in rhesus macaques. We developed a prophylactic vaccine by using a Sendai virus (SeV) vector expressing a single SIV Gag_241-249 CTL epitope fused with enhanced green fluorescent protein (EGFP). Vaccination resulted in induction of SeV-EGFP-specific CD4⁺ T-cell and Gag_241-249-specific CD8⁺ T-cell responses. After a SIV challenge, the vaccinees showed dominant Gag_241-249-specific CD8⁺ T-cell responses with higher effector memory frequencies in the acute phase and exhibited significantly reduced viral loads. These results demonstrate that virus-specific memory CD8⁺ T cells induced by vaccination without virus-specific CD4⁺ T-cell help could indeed facilitate SIV control after virus exposure, indicating the benefit of prophylactic vaccination eliciting virus-specific CTL memory with non-virus-specific CD4⁺ T-cell responses for HIV control.Virus-specific T-cell responses are crucial for controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication (3, 4, 12, 20, 28, 36, 37). Therefore, a great deal of effort has been exerted to develop AIDS vaccines eliciting virus-specific T-cell responses (23, 27, 30, 47), but whether this approach actually results in HIV control remains unclear (1, 6). It is important to determine which T-cell responses need to be induced by prophylactic vaccination for HIV control after virus exposure.Because HIV preferentially infects HIV-specific CD4⁺ T cells (5), induction of HIV-specific memory CD4⁺ T cells by vaccination may increase the target cell pool for HIV infection and could enhance viral replication (42). However, CD4⁺ helper T-cell responses are important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction (11, 40, 43, 46), and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination with non-virus-specific CD4⁺ T-cell help (but without HIV-specific CD4⁺ T-cell help) can exert effective responses after virus exposure. Indeed, the real impact of prophylactic induction of CTL memory itself on HIV replication has not been well documented thus far.We previously developed a prophylactic AIDS vaccine consisting of DNA priming followed by boosting with a recombinant Sendai virus (SeV) vector expressing SIVmac239 Gag (26). Evaluation of this vaccine''s efficacy against a SIVmac239 challenge in Burmese rhesus macaques showed that some vaccinees contained SIV replication whereas unvaccinated animals developed AIDS (15, 27). In particular, vaccination consistently resulted in control of SIV replication in those animals possessing the major histocompatibility complex class I (MHC-I) haplotype 90-120-Ia. Gag_206-216 (IINEEAADWDL) and Gag_241-249 (SSVDEQIQW) epitope-specific CD8⁺ T-cell responses were shown to be involved in SIV control in these vaccinated macaques (14, 16).In the present study, focusing on CD8⁺ T-cell responses directed against one of these epitopes, we have evaluated the efficacy of a vaccine expressing the Gag_241-249 epitope fused with enhanced green fluorescent protein (EGFP) against a SIVmac239 challenge in 90-120-Ia-positive rhesus macaques. The animals exhibited this single-epitope-specific CD8⁺ T-cell response and SeV-EGFP-specific CD4⁺ T-cell responses after vaccination and showed rapid, dominant induction of potent secondary Gag_241-249-specific CD8⁺ T-cell responses after a SIV challenge. Plasma viral loads in these vaccinees were significantly reduced compared to those of naive controls. These results indicate that induction of CD8⁺ T-cell memory without virus-specific CD4⁺ T-cell help by prophylactic vaccination can result in effective CD8⁺ T-cell responses after virus exposure.     

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.     

Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

To investigate the Na⁺-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.The basal body, which is the part of the rotor, is composed of four rings and a rod that penetrates them. Three of these rings, the L, P, and MS rings, are embedded in the outer membrane, peptidoglycan layer and in the inner membrane, respectively (1), while the C-ring of Salmonella species is attached to the cytoplasmic side of the basal body (3). The C-ring is composed of the proteins FliG, FliM, and FliN (25), and genetic evidence indicates that the C-ring is important for flagellar assembly, torque generation, and regulation of rotational direction (33, 34). FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na⁺-type motor of Vibrio alginolyticus (32, 35, 36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30). It has been reported that 33 to 35 copies of FliM assemble into a ring structure (28, 29). FliN contributes mostly to forming the C-ring structure (37). The crystal structure of FliN revealed a hydrophobic patch formed by several well-conserved hydrophobic residues (2). Mutational analysis showed that this patch is important for flagellar assembly and rotational switching (23, 24). The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. Thermotoga maritima FliN exists primarily as a dimer in solution, and T. maritima FliN and FliM together formed a stable FliM₁-FliN₄ complex (2). The spatial distribution of these proteins in the C-ring of Salmonella species was investigated using three-dimensional reconstitution analysis with electron microscopy (28). However, the correct positioning has still not been clarified.The Na⁺-driven motor requires two additional proteins, MotX and MotY, for torque generation (19-21, 22). These proteins form a unique ring structure, the T ring, located below the LP ring in the polar flagellum of V. alginolyticus (9, 26). It has been suggested that MotX interacts with MotY and PomB (11, 27). Unlike peritrichously flagellated Escherichia coli and Salmonella species, V. alginolyticus has two different flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum is used for motility in low-viscosity environments such as seawater (18). As described above, it is driven by a Na⁺-type motor. However, in high-viscosity environments, such as the mucus-coated surfaces of fish bodies, cells induce numerous unsheathed lateral flagella that have H⁺-driven motors (7, 8). We have been focusing on the Na⁺-driven polar flagellar motor, since there are certain advantages to studying its mechanism of torque generation over the H⁺-type motor: sodium motive force can be easily manipulated by controlling the Na⁺ concentration in the medium, and motor rotation can be specifically inhibited using phenamil (10). Moreover, its rotation rate is surprisingly high, up to 1,700 rps (compared to ∼200 rps and ∼300 rps for Salmonella species flagella and E. coli flagella, respectively) (12, 16, 17).Although understanding the C-ring structure and function is essential for clarifying the mechanism of motor rotation, there is no information about the C-ring of the polar flagellar motor of Vibrio species or the flagella of any genus other than Salmonella. Since Vibrio species have all of the genes coding for C-ring components, we would expect its location to be on the cytoplasmic side of the MS ring, as in Salmonella species. In this study, we attempted to isolate the polar flagellar basal body with the C-ring attached and investigate whether it is organized similarly to the H⁺-driven flagellar motor of Salmonella enterica serovar Typhimurium.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.