Catherine Burton

Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy.

Background:HIV-1 infection is characterized by increase in inhibitory receptors and loss of activating receptors on natural killer (NK) cells, resulting in loss of cell activity. Exceptionally, for an inhibitory receptor, the proportion of NK cells bearing CD94-NKG2A decreases during HIV-1 infection. It is not understood whether HIV-1 itself or other concomitant infections drive these changes. Objectives:To investigate the relationship between HIV-1 viraemia and changes in C-type lectin-like receptor expression in NK cells and to investigate the effect of highly active antiretroviral therapy (HAART) on these changes. Methods:Three cohorts of patients were studied: (1) before, during and after treatment interruption in aviraemic and viraemic patients receiving HAART (n = 15); (2) HIV-1-positive treatment-naive individuals (n = 13); and (3) HIV-1-positive individuals receiving successful HAART for a minimum of 1 year without interruption (n = 11). Flow cytometry was used to study the expression of NKG2A before and after treatment interruption and to define expanded populations of NK cells in untreated and treated HIV-1-positive individuals. Assays were performed in vitro to assess the cytotoxicity of the expanded populations. Results:Increases in plasma HIV-1 RNA during treatment interruption in aviraemic HAART-treated individuals did not influence the proportion of NK cells carrying the complex CD94-NKG2A. Loss of NKG2A NK cells corresponded to the dramatic expansion of a distinct population of cells expressing a functional activating CD94-NKG2C receptor with skewed expression of killer cell immunoglobulin-like receptor family and natural cytotoxicity receptors. Conclusion:Changes in the NK cell repertoire during HIV-1 infection were not a result of HIV-1 viraemia alone but resembled those associated with concomitant infections.

A point mutation in CD45 may be associated with an increased risk of HIV-1 infection.

The CD45 antigen is essential for normal antigen receptor-mediated signalling in lymphocytes, and different patterns of splicing of CD45 are associated with distinct functions in lymphocytes. Here we show that abnormal CD45 splicing caused by a C77G transversion in exon A of the gene encoding CD45 (PTPRC) is associated with increased susceptibility to HIV-1 infection.

Reconstitution of CD4+ T cell responses in HIV-1 infected individuals initiating highly active antiretroviral therapy (HAART) is associated with renewed interleukin-2 production and responsiveness

Reconstitution of functional CD4+ T cell responsiveness to in vitro stimuli is associated with continuous highly active antiretroviral therapy (HAART). Thirty‐six antiretroviral naive patients received HAART over 16 weeks. Antigen‐specific, mitogen and interleukin (IL)‐2 induced lymphocyte proliferative responses and specific IL‐2 and IL‐4 production were assessed at each time‐point, together with quantification of HIV‐1 RNA load and lymphocyte populations. Reconstitution of recall responses was limited largely to persistent antigens such as Herpes simplex virus and Candida, rather than to HIV‐1 or neo‐antigens. Recall antigens, mitogens and IL‐2‐induced renewed responses were associated with in‐vitro production of IL‐2, but not IL‐4. Differential responsiveness to low versus high concentration IL‐2 stimulus increases in a stepwise manner, suggesting normalization of IL‐2 receptor expression and improved functionality. These increases in in‐vitro proliferative responses thus probably reflect short lived effector clones, driven by ongoing antigenic stimulus associated with persisting long‐term organisms. In this context non‐responsiveness to HIV‐1 antigens suggests ongoing HIV‐1 specific clonal T cell anergy.

Immune responses and reconstitution in HIV-1 infected individuals: impact of anti-retroviral therapy, cytokines and therapeutic vaccination

Most patients with chronic HIV-1 infection lack functional CD4(+) and CD8(+) HIV-1-specific T cells with proliferative and cytolytic capacity, respectively. This is despite being able to produce intracellular cytokines in response to viral antigens. Protease inhibitor (PI)-based highly active anti-retroviral therapy (HAART) is unable to completely eradicate virus and fails to enable total restoration of immunity including induction of anti-HIV-1 responses. We have taken novel approaches towards the treatment of chronic HIV-1 disease with the aim of instigating long-term non-progressor status and depletion of virus reservoirs. HIV-1-specific CD4(+) and CD8(+) T cell responses were measured following the administration of cytokines, during therapeutic vaccination, and following treatment interruption (TI) or drug therapy change. Administration of cytokines, with or without therapeutic vaccination, in HAART treated patients, improved both CD4(+) and CD8(+) HIV-1-specific T cell responses even in late-stage disease. Virus-specific T cell responses were also seen during TI or when transient viraemia was apparent, and following therapy change from a PI- to a non-nucleoside-based HAART regimen. Reconstitution of HIV-1-specific immune responses was found to be transient and reversal to the previous anergic state was rapid. Viral reservoirs in the latently infected resting CD4(+) T cells, on follicular dendritic cells of germinal centers or even in infected thymic epithelium may be involved in clonal suppression and anergy. These may present major obstacles to the maintenance of HIV-1-specific responses and the eventual eradication of HIV-1.

Expression of PD-L1, a marker of disease status, is not reduced by HAART in aviraemic patients.

Anergy and defective immune responses are characteristic of chronic HIV-1 infection. The programmed death 1 (PD-1)/PD-1 ligand (PD-L1) pathway appears to be involved in downregulating T-cell functionality. We found raised levels of PD-L1 in aviraemic chronically infected HIV-1-positive patients, which may contribute to incomplete immune reconstitution after treatment with HAART.

Thymic Output during Initial Highly Active Antiretroviral Therapy (HAART) and during HAART Supplementation with Interleukin 2 and/or with HIV Type 1 Immunogen (Remune)

The thymic output of patients receiving highly active antiretroviral therapy (HAART) was assessed by sjTREC (signal joint T cell receptor rearrangement excision circle) analysis to determine the thymic contribution to CD4(+) T cell reconstitution during initial therapy and during interleukin 2 (IL-2) and/or Remune supplementation of HAART. Levels of sjTRECs were observed to decline dramatically after the first 4 weeks of HAART and then increased without significant associated changes in CD4(+) T cell counts. HAART supplementation with IL-2 was observed to lead to rapid increases in CD4(+) T cells that were accompanied by sjTREC decreases. No notable changes in CD4(+) T cell counts and sjTRECs were seen in patients receiving HAART supplemented with Remune alone. The results indicate CD4(+) T cell maintenance during initial treatment of HIV-1 with HAART and early CD4(+) T cell reconstitution of patients receiving IL-2 with HAART is largely due to thymus-independent mechanisms, with the thymus making a limited contribution.

The Exon A (C77G) Mutation Is a Common Cause of Abnormal CD45 Splicing in Humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.

Immunological and virological consequences of patient-directed antiretroviral therapy interruption during chronic HIV-1 infection

Increasing numbers of patients are choosing to interrupt highly active antiretroviral therapy (HAART). We describe the effect of patient‐directed treatment interruption (PDTI) on plasma viral loads (pVL), proviral DNA (pDNA), lymphocyte subsets and immune responses in 24 chronically HIV‐1 infected individuals. Patients were divided into group A with pVL > 50 copies/ml and group B with pVL < 50 copies/ml, prior to the PDTI. pVL rose significantly in group B during the first month off HAART and was associated with a significant decrease in CD4 T‐cell count. At baseline there was a significant difference in HIV‐1 pDNA levels between groups A and B, however, levels significantly increased in group B, but not in group A during PDTI becoming equivalent after 1 month PDTI. We have previously shown no increase in pDNA over the time of substitution in patients switching HAART regimens despite a small rebound in pVL. These observations indicate that to protect low pDNA levels PDTI should be discouraged and that changing regimen at the first sign of failure should be advised where possible. Only transient, no longer than 4 week, HIV‐1‐specific responses were observed during PDTI in 5/24 patients, 2 from group A and 3 from group B. The low numbers of responders and the transient nature of the anti‐HIV‐1 immune responses do not favour the auto‐vaccination hypothesis.

Combined use of cytokines, hormones and therapeutic vaccines during effective antiretroviral therapy

Immune-based therapies using vaccines, cytokines and hormones are being considered in the context of effective antiretroviral therapy to induce immunologically defined long-term nonprogressor status in chronically infected HIV-1 patients. Such immunotherapy must allow induction or regeneration of anti-HIV-1 immune responses with the potential to control viremia, activate and eradicate viral reservoirs, and alleviate the immunosuppression caused by HIV-1, eventually possibly reaching the status of a virologically defined ‘elite controller’ with an absence of detectable viremia and no progression to disease over a long period of time. This article summarizes pilot studies utilizing therapeutic vaccines, cytokines and/or hormones in treated HIV-1 infection, and focuses on novel agents and immunotherapeutic options that may have the potential to augment or replace existing antiretroviral therapy with the aim of inducing nonprogressor status in the infected host.

Impact of NNRTI compared to PI-based highly active antiretroviral therapy on CCR5 receptor expression, β-chemokines and IL-16 secretion in HIV-1 infection

Summary Interleukin‐16 (IL‐16) and the β‐chemokines (RANTES, monocyte chemotactic protein‐1 (MCP‐1), macrophage inhibitory protein (MIP)‐1α and (MIP)‐1β) are soluble in vitro suppressors of macrophage tropic HIV‐1 strains. The reduction of HIV‐1 RNA plasma levels in late‐stage patients receiving protease inhibitors has been associated with increased concentrations of MIP‐1α, MIP‐1β, RANTES and IL‐16 and a decrease in levels of MCP‐1. We determined plasma levels of MCP‐1, MIP‐1α, MIP‐1β, RANTES and IL‐16 during the first 16 weeks of highly active antiretroviral therapy (HAART) in chronic HIV‐1‐infected patients. Patients were administered one of two therapeutic regimens based on either   a   protease   inhibitor (PI) or   a   non‐nucleoside   reverse   transcriptase    inhibitor (NNRTI).   No differences were seen in the levels of RANTES and IL‐16 over the first 16 weeks of HAART in either treatment group. MCP‐1 decreased significantly in the PI‐treated group over the first 16 weeks of HAART (P = 0·0003). A significant increase was observed in the levels of MIP‐1α and MIP‐1β in the NNRTI cohort (P = 0·0010 and P = 0·0012, respectively). A significant decrease in levels of MIP‐1α and MIP‐1β (P = 0·0015 and P = 0·0299, respectively) was observed over the 16 weeks in the PI cohort. A significant difference was seen when the levels of MIP‐1α and MIP‐1β were compared between the NNRTI and the PI cohorts at week 16 (P = 0·04 and P = 0·05, respectively). Evaluation of CCR5 expression ex vivo revealed no difference between the two treatment groups. Patients were genotyped for CCR5 Δ32 and the incidence of heterozygosity was lower than in the HIV‐1 seronegative controls (3% compared to 19%).