New observations on CD8 cell responses (2003)

Introduction Most HIV vaccine development programs target the induction of strong, broad, high-affinity HIV-specific CD8 T-cell responses directed against several HIV antigens. Over the past year, some initial immunogenicity results were reported from human trials. In the first preventative HIV vaccine study to be carried out in Africa, 40 HIV-seronegative Ugandan volunteers were randomly assigned to receive a canarypox vector containing HIV-1 clade B (env and gag-pro) antigens (ALVAC-HIV), control vector containing the rabies virus glycoprotein G gene, or saline placebo. Cytotoxic T lymphocyte (CTL) activity against target cells expressing clade A, B, and D antigens was assessed using chromium-release and enzyme-linked immunospot (ELISPOT) assays. Twenty per cent of vaccine recipients generated detectable cytolytic responses to either Gag or Env, and 45% had vaccine-induced HIV-specific CD8 T-cell responses, as measured using the ELISPOT assay. Although the immunogenicity of this clade B-based vaccine was low, ALVAC-HIV elicited CD8 T-cell responses with detectable cross-reactivity against clade A and D antigens in a significant proportion of vaccine recipients [1]. In another study using ALVAC-HIV vaccine with or without rgp120/SF-2 [2], HIV-1-specific CTL were detected in the blood of 71% and, importantly, in the rectum of 57% of the volunteers. This shows that parenteral administration of this vaccine candidate can lead to the presence of HIV-specific T cells at a mucosal site. Over the next year anticipated results from trials involving adenovirus vectors and modified vaccine Ankara will be announced. Other recent reports of early phase candidate vaccines are not so promising. In one study of a DNA vaccine [3], no CD8 T-cell responses were generated in human volunteers. Therapeutic immunization approaches are also currently under active development, and new information on the role of different antigen presenting cells may lead to better immunogenicity. In one study, plasmacytoid dendritic cells were able to activate influenza-specific T cells in vitro. Plasmacytoid dendritic cells were compared with CD11c+ dendritic cells, the most potent antigen-presenting cells, for their capacity to activate T-cell responses. After influenza virus exposure, both dendritic cell types showed an equivalent efficiency to expand anti-influenza virus CTL [4]. Other agents that could be used to potentiate virus-specific CD8 T cells are under active investigation. Over the past year, it was reported that thalidomide analogues have potent co-stimulatory activity for virus-specific CD8 T cells, and thus could help function as a novel immune-adjuvant therapy [5]. In addition, structured treatment interruption was shown in a large Swiss Spanish study to boost HIV-specific CD8 T-cell responses [6]. A recent report of apparent HIV-1 superinfection, despite the presence of strong and broad CD8 T-cell responses [7], has caused significant concern with regard to vaccines reliant on inducing CD8 T-cell responses. Altfeld and colleagues [7] reported on the sudden breakthrough of plasma viremia that occurred after prolonged immune containment in an individual infected with HIV-1, at a time when 25 distinct CD8 T-cell epitopes in the viral proteins Gag, reverse transcriptase, integrase, Env, Nef, Vpr, Vif and Rev were being targeted. Sequencing of the virus in the plasma and cells showed that superinfection with a second clade B virus was coincident with the loss of immune control. These data showed that HIV-1 superinfection can occur in the setting of a strong and broadly directed virus-specific CD8 T-cell response. This has focused attention on the quality of HIV-specific responses elicited after HIV-1 infection. It is possible that only a proportion of CD8 T-cell responses may exert significant antiviral pressure (`driver responses'), in contrast to those that may exert weak pressure on the virus (`passenger responses') [8]. The nature of the T-cell response elicited to each epitope needs to be taken into account in vaccine or immunotherapeutic design. Differentiation of CD8 cells and HIV infection Functional CD8 T cells proliferate and mediate antiviral activity after the recognition of cognate antigen. When naive CD8 T cells are activated during a primary infection, they undergo functional and phenotypic changes to fulfil these functions and differentiate into effector cells [9]. After clearance of the virus, there is a contraction of the immune response in which most of the effector cells die, and a small population of mature antigen-specific memory cells persist. In chronic HIV infection, the immune system fails to contain viral replication, and the continuous presence of antigen leads to persistent immune activation, with skewed consequences for CD8 T-cell differentiation and maturation [10,11]. Several models for CD8 T-cell differentiation have been described using cell surface markers [12–14]. HIV-specific CD8 T cells have features of an intermediate stage of differentiation, with low levels of perforin expression and reduced cytolytic capacity compared with cytomegalovirus-specific CD8 T cells. However, recent evidence suggests that reduced perforin expression and the loss of cytotoxic activity is a feature of chronic viral infections in general [15–19], and may represent redistribution effects between the peripheral circulation and sites of viral replication. In long-term non-progressors, perforin expression coupled with proliferation appears to play a protective role [20], whereas in more advanced patients, the HIV-specific T cells have lost their proliferative capacity. The loss of proliferative capacity correlates with the expression of CD57 [21], again a phenomenon observed in other viral infections [22,23]. New insights into CD8 T-cell responses in HIV-infected children Vertical transmission from mother to infant continues to be a major problem in many parts of the world, and this mode of transmission is likely to pose particular challenges as the immune system of the fetus and infant is not fully mature. The past year has seen considerable increases in the knowledge about the function of CD8 T cells in HIV-infected pediatric patients. Infection of the thymus causes local IFN-α secretion, which in turn leads to MHC class I upregulation [24]. This correlates with the downregulation of CD8 on developing CD8 T cells in the thymus and a concomitant gradual loss of responsiveness [25]. This has broad implications for the functionality of CD8 T cells in pediatric HIV infection, as an active thymus can re-stock the peripheral immune system. The importance of the thymus in children was emphasized by the observation that the initiation of highly active antiretroviral therapy results in the rapid restoration of T-cell receptor CDR3 β repertoire diversity in the CD45RA positive CD8 T-cell subset, indicative of naive cell replenishment by thymic emigrants [26]. A comprehensive study of pediatric CD8 T-cell responses to HIV-1 [27] found that the response is influenced, quantitatively and qualitatively, by the individual's age and CD4 T-cell phenotype and counts. Pediatric patients needed to be older than 3 years of age and stay above a minimum number of circulating CD4 T cells to mount strong and antigen-driven CD8 T-cell responses against HIV. When these conditions were not met, CD8 T-cell responses were poor and the HIV load was high. These two observations may at first appear to be contradictory, but may be explained by the following model: under the lack of CD4 T-cell help or the influence of as yet undefined young age-dependent negative factors, CD8 T cells are unable to respond efficiently to HIV, and cannot contribute to the lowering of the viral burden. In contrast, under circumstances in which CD4 T-cell help is present, and the negative influence of young age is absent, HIV-specific CD8 T cells can respond in an antigen dose-dependent manner, and contribute to eliminating virus and lowering the HIV load. This model may also help reconcile the apparent inconsistencies in the literature in which some studies have described an inverse correlation between the numbers of circulating HIV Gag-specific CD8 T cells and the HIV load, suggesting immune control of the virus [28]. However, subsequent studies have observed either no correlation [29–31], or a direct positive correlation between CD8 T-cell responses and viral load [32]. This confusion has continued over the past year, with three studies reporting inverse correlations [33–35], another no correlation [36], and a third study reporting no or a direct positive correlation between CD8 T-cell responses to HIV and the viral load [37]. The latter study reached different conclusions depending on the method used for the detection of responses, and this may explain some of the conflicting data on this matter. HIV is the antigen that drives the CD8 T-cell response, the target virus for HIV-specific CD8 T-cell responses, and the cause of the generalized immunosuppression and chronic immune activation that over time disrupts the ability to mount efficient responses. The correlation between viral load and CD8 cell response will thus depend on the patient cohort studied. An inverse correlation may be observed if many of the study subjects have retained the ability to mount effective CD8 T-cell responses to the virus. On the other hand, a direct positive correlation may result when the immune system of study subjects deteriorates and the ability of CD8 T-cell responses to suppress viral replication wanes. Antiviral factors produced by CD8 T cells Various groups continue to work towards the identification of factor(s) produced by CD8 T cells that have antiviral activities. One study showed that certain protease inhibitors, particularly leupeptin, could block anti-HIV activity in CD8 cell culture fluids as well as inhibit CD8 cell non-cytotoxic antiviral response [38]. The pretreatment of CD8 cells with leupeptin reduced the non-cytotoxic antiviral response, further supporting an inhibitory effect on a CD8 cell product. These results suggest that a part of the CD8 cell non-cytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. In a controversial study [39], another group identified human α-defensins 1, 2, and 3 as proteins secreted from CD8 T cells from long-term non-progressors with powerful anti-HIV-1 activity. They suggested that α-defensins are the major component of CD8 cell antiviral factor activity. However, a very recent study showed that CD8 cell antiviral factor-mediated HIV inhibition is distinct from α-defensin HIV inhibition [40], indicating that CD8 cell antiviral factor may result from several distinct factors. Antiretroviral drugs and CD8 T cells Both HIV-specific CD8 T cells and antiretroviral drugs exert selection pressures on HIV. In a new study [41], these dual selective pressures have been observed to act on the same region of HIV protease. A new human leukocyte antigen (HLA)-A2 restricted CTL epitope in HIV protease was mapped to amino acids 76–84 [PR82V(76–84)]. This epitope includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among HLA- A2-positive HIV-infected patients who had been treated with protease inhibitors (and who had developed resistance to the protease inhibitors), the wild-type PR82V(76–84) epitope was commonly recognized by CTL. In contrast, the mutant PR82A(76–84) epitope was generally not recognized by wild-type-specific CTL, suggesting that the protease inhibitor-selected V82A mutation can act both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8 T-cell response to the wild-type virus sequence. These results show that both HIV-specific CD8 T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution. The importance of evolutionary pressures from antigen-specific T-cell immunity was reinforced by a study that analysed HIV reverse transcriptase sequences from a large HLA-diverse population of HIV-1-infected individuals [42]. Polymorphisms in HIV-1 were most evident at sites of least functional or structural constraint, and were frequently associated with particular host HLA class I alleles. The absence of polymorphism was also HLA allele-specific, and at a population level, the degree of HLA-associated selection in the viral sequence was predictive of the viral load. These results support a fundamental role for HLA-restricted immune responses in driving and shaping HIV-1 evolution in vivo. How HLA-restricted T-cell responses do this is an area of active investigation. One possibility may be that HLA may be linked closely with transporter-associated antigen processing genes [43]. Epitope-specific influences may be highly specific. The differential ability of HLA-B*5701 long-term non-progressors and progressors to restrict HIV replication appears not to be caused by the loss of recognition of autologous viral gag sequences [44]. HIV epitopes recognized by HIV-specific CD8 T cells Several studies have comprehensively surveyed HIV-specific CD8 T-cell responses using large panels of peptides [7,36,45–47]. A study in primary HIV infection showed that the earliest responses detected were directed against HIV Nef, Tat, Vpr and Env [46]. The use of autologous virus-derived sequence peptides in T-cell assays has helped identify important discrepancies from results obtained from consensus sequences. One study compared the CD8 T-cell response to CD4 T-cell targets infected with either laboratory-adapted HIV(IIIB) or the patient's own virus [47]. The magnitude of the IFN-γ response declined with disease progression, especially to autologous virus. Therefore, in advanced-stage patients, viral-specific CD8 T cells recognizing consensus epitopes persist from an earlier response but no longer effectively recognize autologous virus. Mucosal tissues and HIV-specific CD8 T cells Whereas most studies on HIVspecific CD8 T cells have been carried out using peripheral blood, a few studies have accessed other tissues. HIV-specific CD8 T cells have been identified in breast milk [48], and their presence suggests a role in limiting the transmission of virus from mother to infant. The specificity of HIV-specific CD8 T cells in mucosa and peripheral blood were shown largely to overlap [2]. Improved methods to isolate T cells from gut-associated lymphoid tissues [49] have helped generate new data on the function and frequency of HIV-specific CD8 T cells in gut-associated lymphoid tissues. The frequency of HIV Gag (SL9)-specific CD8 T cells was increased in gut-associated lymphoid tissues relative to peripheral blood mononuclear cells by up to fivefold [50].