aMRC Human Immunology Unit and Oxford NIHR Biomedical Research Centre, Weatherall Institute of Molecular Medicine John Radcliffe Hospital, Oxford OX3 9DS, UK
bThe Jenner Institute, University of Oxford, Compton, Newbury, Berkshire RG20 7NN, UK
cCentre for Vascular Research, University of New South Wales, NSW, Australia.
Received 31 March, 2009
Revised 22 July, 2009
Accepted 6 August, 2009
Correspondence to Dr Lucy Dorrell, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK. Tel: +44 1865 222145; fax: +44 1865 222502; e-mail: firstname.lastname@example.org
The goal of therapeutic immunization in HIV-1 infection is to limit dependence on highly active antiretroviral therapy (HAART). Therapeutic vaccines that boost AIDS virus-specific T cell responses in simian immunodeficiency virus (SIV)-infected macaques can augment immune control in chronic infection, but clinical trials in HIV-1-infected individuals have generally been unsuccessful [1–5]. This could reflect differences in the magnitude or quality of the vaccine-boosted responses in humans and monkeys or in the challenge viruses. Murine models of chronic virus infections indicate that virus-induced immunosuppression mediated by interleukin (IL)-10 and/or programmed death-1 signalling can limit the efficacy of immune responses induced by either natural infection or therapeutic vaccination [6,7]. We previously evaluated a therapeutic vaccine comprising an HIV-1 Gag/multiepitope immunogen vectored by modified vaccinia virus Ankara (MVA.HIVA) in HAART-treated patients [8,9] (Fig. 1a). Although MVA.HIVA vaccination augmented functional virus-specific CD8+ T cells, we wished to investigate whether an IL-10 response was induced and whether this influenced the magnitude of the cellular response to the HIV-1 immunogen. Plasma IL-10 concentrations were determined retrospectively by ELISA in a blinded analysis: baseline values were similar to those of 11 HIV-uninfected controls (means 3.4 and 4.5 pg/ml, respectively, P = 0.34). Postvaccination, IL-10 levels were not significantly increased, although elevations exceeding the mean +2 SD of HIV-uninfected control values were observed in three patients (Fig. 1b). There was no correlation between IL-10 increment and change in frequency of HIV-1-specific T cells (data not shown).
We extended the study by scheduling a third MVA.HIVA vaccination (1 × 108 pfu, given by intradermal injection in the deltoid region) to be followed by an analytical therapy interruption (ATI) 7 days later, in order to assess the capacity of vaccine-boosted CD8+ T cells to limit viral rebound (schema shown in Fig. 1a). Principal eligibility criteria for re-vaccination were the same as for study entry, that is CD4 cell count greater than 300 cells/μl and plasma viral load (pVL) less than 50 copies/ml for at least 12 months. Criteria for resuming HAART after interruption were: two consecutive CD4 cell counts less than 300 cells/μl or plasma HIV-1 RNA levels greater than 105 copies/ml or HIV-related symptoms. The study was approved by the relevant UK ethical and regulatory agencies and was conducted in accordance with ICH-GCP requirements. Patients provided their written informed consent.
Three patients (patients 001, 013 and 021) received a third MVA.HIVA vaccination 15–30 months after their first dose and interrupted therapy as planned. In all three, pVL became detectable by day 14 and had risen to more than 105 copies/ml by day 28 (Fig. 1a, c). Soon after, all met criteria for resuming HAART. They subsequently achieved virological re-suppression and no serious adverse events occurred during the ATI. Unexpectedly, CD8+ T cell responses to previously targeted MVA.HIVA epitopes did not increase until day 28, consistent with a response to viraemia rather than vaccination (data not shown). As no cause for the lack of cellular responses to the third vaccine dose was identified, the study was halted. In all three patients, plasma IL-10 concentrations increased during ATI and were strongly correlated with pVL (Spearman r = 0.584; P = 0.005). Of note, IL-10 levels were not elevated in these three individuals after VACs 1 and 2 (Fig. 1b).
Brockman et al.  recently reported up-regulation of IL-10 in diverse peripheral blood mononuclear cell subsets during uncontrolled HIV-1 viraemia. Therefore, we analysed the cellular origin of IL-10 in vaccinees who either continued or interrupted HAART. Using a cytokine capture assay with an enrichment step to enable quantification of rare IL-10-secreting cells, we detected constitutive IL-10 secretion in patients continuing HAART, particularly by monocytes and B cells, but no HIV-1-specific IL-10 production in any cell type (Fig. 1d). In contrast, in patients undergoing ATI, a population of circulating HIV-1-specific IL-10-secreting cells expanded within the CD4+ T cell subset, in tandem with changes in their plasma IL-10 levels; HIV-1-specific IL-10 secretion was only detected within the CD8+ T cell subset in patient 021 on day 14 (Fig. 1e).
As IL-10 antagonizes Th1 responses , we next investigated the effect of IL-10 blockade on the ex-vivo proliferative capacity of HIV-1-specific T cells sampled from vaccinees remaining on HAART or undergoing ATI. IL-10-neutralizing and receptor blocking antibodies significantly augmented virus-specific T cell responses in 10 HAART-suppressed patients but failed to restore the proliferative capacity of T cells sampled during the ATI (Fig. 1f). This contrasted with the study by Brockman et al. , which showed that IL-10 blockade augmented the proliferative and cytokine-secreting capacity of HIV-1-specific T cells in viraemic, but not HAART-suppressed individuals. These observations may indicate differences in the reversibility of T cell dysfunction among acutely and chronically viraemic individuals and/or the combined effects of IL-10 neutralizing and receptor-blocking antibodies.
Elevations in IL-10 have been reported to accompany rising plasma viraemia during primary HIV-1 infection and after HAART withdrawal in patients who initiated therapy during primary infection [12–14]. This may reflect a host regulatory response to immune activation induced by HIV-1. Sample limitations precluded further evaluation of the phenotype of the HIV-1-specific IL-10-secreting CD4+ T cells detected in the patients we studied. However, HIV-1-specific IL-10-producing T cells with suppressive capacity have been recently identified in chronically infected individuals . Nonhuman primate models suggest that very early immunoregulatory responses to SIV may protect against AIDS but could be counterproductive if too late to limit immune activation, yet early enough to impede adaptive T cell responses [16,17]. Modulation of these responses might therefore enhance immune control in the setting of chronic infection. However, before considering strategies to manipulate the IL-10 pathway, further clarification is needed regarding the immunosuppressive capacity of constitutive and virus-specific IL-10-producing cell populations under viraemic and aviraemic conditions. Furthermore, simultaneous blockade of other signals (e.g. the interaction of PD-1 with its ligand PD-L1) may be necessary for full restoration of antiviral functions in CD8+ T cells [7,18]. Addressing these questions may be key to the development of effective T cell-based vaccination strategies for HIV-1 control.
We thank the patients who participated in this study. We are grateful to Dr Anne Bridgeman, MRC Human Immunology Unit, for QC analysis of the MVA.HIVA lot used in this study. This work was funded by Medical Research Council UK, NIHR Biomedical Research Centre Programme UK, the Jenner Institute, UK (PB, LD, TH, AMcM) and NHMRC, Australia (MPD).
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