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).     

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.     

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.     

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.     

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.     

Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8⁺ T-lymphocyte responses in Mamu-A*01⁺ rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01⁺ rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8⁺ T-lymphocyte response in the absence of secondary Gag p11C-specific CD8⁺ T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8⁺ T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8⁺ T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.CD8⁺ T lymphocyte responses play a central role in controlling human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) infections in nonhuman primates (18, 20, 29, 41). Naturally occurring virus-specific CD8⁺ T-lymphocyte responses typically focus on a limited number of dominant epitopes (52). However, accumulating data indicate that a broad cellular immune response, in which multiple viral epitopes are recognized by CD8⁺ T lymphocytes, confers better protection against viral replication than a restricted cellular immune response (26, 33). Therefore, it has been suggested that increasing the magnitude of subdominant epitope-specific responses may increase the breadth of a cellular immune response and provide enhanced protection against HIV/SIV replication.An understanding of the factors that influence the immunodominance hierarchy of viral epitopes will be needed to develop vaccination strategies that can generate the greatest breadth of virus-specific CD8⁺ T-lymphocyte responses. Differences in antigen processing, competition between epitope peptides for major histocompatibility complex (MHC) class I molecules, T-cell receptor (TCR) repertoire, TCR affinity for peptide class I complexes, and immunodomination have been shown to contribute to the dominance of an epitope-specific response (6, 10, 24, 32, 45, 52). In addition, studies have shown that immunodominance patterns for T-lymphocyte epitopes may differ following a primary and secondary exposure to the same viral antigen (4, 5, 43).In the present study, we observed that Mamu-A*01⁺ rhesus monkeys primed with plasmid DNA and boosted with recombinant adenovirus (rAd) vaccines encoding SIVmac239 Gag-Pol-Nef and HIV-1 Env proteins generated Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses of comparable magnitude. However, while there was a significant expansion of Gag p11C-specific CD8⁺ T-lymphocyte populations following challenge with pathogenic simian-human immunodeficiency virus 89.6P (SHIV-89.6P), there was no significant expansion of the Env p41A-specific CD8⁺ T-lymphocyte populations. We hypothesized that factors influencing the relative immunodominance of the primed Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses after viral challenge may have contributed to the observed differences in their secondary expansion. In the present study, we sought to identify the potential factors contributing to this immunodominance.     

Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection

Rapid depletion of memory CD4⁺ T cells and delayed induction of neutralizing antibody (NAb) responses are characteristics of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Although it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV replication, a recent study has shown that a single passive NAb immunization of rhesus macaques 1 week after SIV challenge can result in reduction of viral loads at the set point, indicating a possible contribution of postinfection NAb responses to virus control. However, the mechanism accounting for this NAb-triggered SIV control has remained unclear. Here, we report rapid induction of virus-specific polyfunctional T-cell responses after the passive NAb immunization postinfection. Analysis of SIV Gag-specific responses of gamma interferon, tumor necrosis factor alpha, interleukin-2, macrophage inflammatory protein 1β, and CD107a revealed that the polyfunctionality of Gag-specific CD4⁺ T cells, as defined by the multiplicity of these responses, was markedly elevated in the acute phase in NAb-immunized animals. In the chronic phase, despite the absence of detectable NAbs, virus control was maintained, accompanied by polyfunctional Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered virus control, suggesting possible synergism between NAbs and T cells for control of HIV/SIV replication.Virus-specific CD4⁺ and CD8⁺ T-cell responses are crucial for the control of pathogenic human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections (5, 6, 20, 23, 30, 39, 40). However, CD4⁺ T cells, especially CCR5⁺ memory CD4⁺ T cells, are themselves targets for these viruses, which may be an obstacle to potent virus-specific CD4⁺ T-cell induction (10, 47, 52). Indeed, HIV-1/SIV infection causes rapid, massive depletion of memory CD4⁺ T cells (26, 31), and host immune responses fail to contain viral replication and allow persistent chronic infection, although virus-specific CD8⁺ T-cell responses exert suppressive pressure on viral replication (15).Recently, the importance of T-cell quality in virus containment has been high-lighted, and T-cell polyfunctionality, which is defined by their multiplicity of antigen-specific cytokine production, has been analyzed as an indicator of T-cell quality (4, 8, 11, 41). However, there has been no evidence indicating an association of polyfunctional T-cell responses in the acute phase with HIV-1/SIV control. Even in the chronic phase, whether polyfunctional CD4⁺ T-cell responses may be associated with virus control has been unclear, although an inverse correlation between polyfunctional CD8⁺ T-cell responses and viral loads has been shown in HIV-1-infected individuals (4).Another characteristic of HIV-1/SIV infections is the absence of potent neutralizing antibody (NAb) induction during the acute phase (7). This is mainly due to the unusually neutralization-resistant nature of the virus, such as masking of target epitopes in viral envelope proteins (24). Whether this lack of effective NAb response contributes to the failure to control the virus, and whether NAb induction in the acute phase can contribute to virus control, remains unclear. Previous studies documenting virus escape from NAb recognition suggested that NAbs can also exert selective pressure on viral replication to a certain extent (38, 45, 49), but it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV-1/SIV replication (34, 37).By passive NAb immunization of rhesus macaques after SIV challenge, we recently provided evidence indicating that the presence of NAbs during the acute phase can result in SIV control (50). In that study, passive NAb immunization 1 week after SIVmac239 challenge resulted in transient detectable NAb responses followed by reduction in set point viral loads compared to unimmunized macaques. However, the mechanism of this virus control has remained unclear. In the present study, we found rapid appearance of polyfunctional Gag-specific CD4⁺ T-cell responses after such passive NAb immunization postinfection. These animals maintained virus control for more than 1 year in the absence of detectable plasma NAbs, which was accompanied by potent Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered primary and long-term SIV control.     

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.     

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.     

Human Immunodeficiency Virus Type 1 Escapes from Interleukin-2-Producing CD4+ T-Cell Responses without High-Frequency Fixation of Mutations

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4⁺ T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4⁺ T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4⁺ T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to >1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4⁺ T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4⁺ T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4⁺ T cells rather than to the fixation of escape mutations at high frequencies.In the typical course of acute human immunodeficiency virus type 1 (HIV-1) infection an initial burst of high-level viremia is reduced by at least 100-fold to a set point level (11, 12). This precipitous drop in viral load is suggestive of a partially effective host immune response to primary HIV-1 infection. Several lines of evidence support an important role for CD8⁺ T cells in suppressing HIV-1 replication in acute infection: principally, the decline in HIV-1 viremia is temporally associated with the emergence of an HIV-1-specific CD8⁺ T-cell response, and the in vivo depletion of CD8⁺ T cells in simian immunodeficiency virus-infected macaques consistently results in elevated viral loads (7, 24, 30). Consistent with the application of effective immune pressure, it has been well established that HIV-1- and simian immunodeficiency virus-specific CD8⁺ T cells drive the emergence and fixation of escape mutations in the epitopes that they target (1, 3, 8, 18, 31, 33, 34). This evidence has contributed to the prioritization of vaccine candidates that elicit potent HIV-1-specific CD8⁺ T-cell responses.The role of CD4⁺ T-cell responses in the response to acute HIV-1 infection is less clear. There is compelling evidence that CD4⁺ T-cell help may be critical for the establishment of a qualitatively and quantitatively robust CD8⁺ T-cell memory pool for persistent virus infections (4, 9, 17, 37, 39). Furthermore, an important role for CD4⁺ help in maintaining an effective CD8⁺ T-cell response has been established in the lymphocytic choriomeningitis virus model of chronic viral infection (28, 45). Evidence in support of a role for the CD4⁺ T-cell response to HIV-1 infection in suppressing viral replication is derived from studies which demonstrated that a CD4⁺ T-cell response characterized by vigorous proliferation and production of interleukin-2 (IL-2) is associated with control of viremia (6, 35). It has further been demonstrated that the functional defect of CD8⁺ T cells observed in chronic HIV-1 infection can be induced in vitro by the depletion of CD4⁺ T cells or the addition of IL-2-neutralizing antibodies and can be corrected in vivo by vaccine-mediated augmentation of HIV-1-specific CD4⁺ T-cell responses (26). These observations have suggested that an IL-2-producing response may be necessary for controlling viremia. However, in the majority of HIV-1-infected individuals, a qualitative impairment of the HIV-1-specific CD4⁺ T-cell response occurs early after infection, resulting in the loss of proliferative capacity as well as the ability to produce IL-2 (43). This impairment correlates well with levels of antigen and viremia (29). The relationship between viral control and the presence of IL-2-producing HIV-specific CD4⁺ T-cell responses must be interpreted with caution, however, as the causal relationship between these two factors is unclear. The maintenance of an IL-2-producing HIV-1-specific CD4⁺ T-cell proliferative response could simply be the result of control of viremia achieved through another means, rather than causal in the association. Therapeutic administration of IL-2 to chronically infected individuals failed to reveal any clinical benefit, perhaps supporting that IL-2 is a marker, rather than a driver, of immunological control (25). However, it is unclear whether the systemic administration of IL-2 effectively substitutes for the targeted production of IL-2 by HIV-1-specific CD4⁺ T cells.The fixation of escape mutations in CD4⁺ T-cell epitopes during acute infection would provide direct evidence that CD4⁺ T cells apply immunological pressure against HIV-1. Harcourt et al. identified epitopes targeted by proliferative CD4⁺ T-cell responses in chronically infected individuals and sequenced these epitopes from proviral DNA at multiple time points (16). Variations in these epitope sequences were observed over time, and a minority of these variants failed to stimulate CD4⁺ T-cell lines raised against the index peptide. This study indicated the potential for HIV-1 virus to escape within proviral populations. However, the observation that the majority of emergent variants were still able to stimulate CD4⁺ T-cell responses argues against potent selective pressure for escape mutants (16). A second study examined gamma interferon (IFN-γ)-producing CD4⁺ T-cell responses and contemporaneous circulating virus epitopes in a cohort of chronically infected, untreated, HIV-1-infected individuals. A lack of intrapatient variability within CD4⁺ T-cell epitopes was observed in this study, and while two of four subjects exhibited epitope sequences that differed from the consensus HIV-1 sequence, there was a trend to greater sequence variability outside of epitopic regions, arguing against potent immune pressure (23). These studies support that HIV-1-specific CD4⁺ T-cell responses fail to exert potent selective pressure against cognate epitopes in chronic infection; however, it is difficult to determine whether or not the observed epitopic variations are indicative of relatively weak selective pressures. Since the overall cellular immune response to HIV-1 infection is particularly robust and effective during the acute phase of infection, we examined the kinetics of the HIV-1-specific IL-2-secreting CD4⁺ T-cell-mediated immune response during acute/early HIV-1 infection and studied the effects of this response on circulating plasma viruses.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

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.     

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

CD4⁺ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4⁺ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4⁺ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.The development of an effective vaccine against HIV has been hampered by an incomplete understanding of the correlates of protection against the virus. It is generally accepted that a robust antibody response and cytotoxic T-lymphocyte (CTL) response are required to control the disease and to prevent progression to AIDS (2, 17, 19, 20, 36, 38-42). Both of these arms of the immune system require help from CD4⁺ helper T cells (1, 27, 48). However, several important aspects of the CD4⁺ helper T-cell response remain poorly defined; these include the factors that determine epitope immunodominance in the CD4⁺ T-cell response, the relationship of specificity in the CD4⁺ T-cell response to specificity in the antibody and CD8⁺ responses, and the investment made by HIV (or any pathogen) to control the CD4⁺ T-cell response.Previous studies of mice showed that antigen structure modulates antigen processing and presentation of CD4⁺ helper T-cell epitopes (3-6, 9, 10, 23, 24, 43). Immunodominant CD4⁺ helper T-cell epitopes raised in response to immunization with the HIV envelope glycoprotein gp120 were found adjacent to flexible loops between elements of secondary structure (10). This was rationalized by the fact that flexible loops more readily conform to protease active sites and therefore are preferentially cleaved by proteases during antigen processing (10, 14, 15). Helper T-cell epitopes of gp120 in humans infected with HIV were also found flanking flexible loops (30). Dominant epitopes were located in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops (30). These results suggested that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in both mice and humans.In reviewing previous studies mapping the helper T-cell response to gp120, we noted a marked absence of CD4⁺ T-cell responses to regions of the outer domain that coincided with the locations of highly conserved disulfide bonds (Fig. ​(Fig.1).1). Disulfide bonds have previously been shown to interfere with presentation of nearby helper T-cell epitopes (13, 26). Thus, we hypothesized that disulfide bonds stabilized these regions of the protein, protecting them from proteolysis. This resulted in the exclusion of these regions from presentation to helper T cells. We further hypothesized that the deletion of these disulfide bonds would result in the production of new helper T-cell epitopes by creating localized regions of flexibility that could now be processed and presented to T cells. The creation of new helper T-cell epitopes could also potentially lead to changes in the antibody response.Open in a separate windowFIG. 1.Gaps in helper T-cell epitope frequency in the outer domain of HIV gp120 coincide with the locations of disulfide bonds. The graph illustrates the frequencies of responses by residue for the combined profiles from immunized BALB/c and CBA mice (gray area) and for a group of seven HIV-infected human subjects (black line) (10, 30).For the present work, we constructed three disulfide-bond variants of gp120 by replacing paired cysteines in the outer domain with alanines. Characterization of the variants revealed that the proteins were structurally distinct from one another and from wild-type gp120. Groups of 10 BALB/c mice immunized with these proteins produced patterns of helper T-cell responses that were very different from each other and from that of a group of 10 BALB/c mice immunized with wild-type gp120. In general, the T-cell response was reduced in mice immunized with the variant proteins. For one of the variants, anti-gp120 antibody titers were increased and exhibited CD4-blocking activity.