Although the immune response to human immunodeficiency virus (HIV) cannot eradicate the virus, vigorous HIV-specific CD4+ T-helper and CD8+ cytotoxic T-lymphocyte responses are associated with viral control and long-term nonprogression [1,2]. Highly active antiretroviral therapy (HAART) reduces morbidity and mortality without virus elimination or effective immune control of HIV-1 . Immunotherapy strategies that enhance HIV-1-suppressive immune responses are needed in order to complement, reduce or even possibly replace HAART.
Dendritic cells are potent cellular adjuvants for a therapeutic HIV-1 vaccine. Although HIV-1 infection can adversely affect dendritic cell function, monocyte-derived dendritic cells from patients with HIV-1 infection remain mostly uninfected and functionally intact [4,5]. Dendritic cells generated ex vivo from peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients and electroporated with mRNA encoding Gag elicit strongly responsive CD4+ and CD8+ T cells in vitro[6,7]. Immunotherapy with autologous dendritic cells loaded with inactivated HIV  has resulted in favorable clinical and immunological responses. However, virus inactivation is difficult to standardize . We have developed a highly efficient, transient and clinically safe electroporation procedure to introduce mRNA into dendritic cells , resulting in efficient antigen presentation . Using a similar strategy, Routy et al. reported on vaccination of HIV-1-infected individuals with HIV mRNA-electroporated dendritic cells, resulting in proliferative T-cell responses.
The regulatory HIV proteins Tat, Rev and Nef are expressed during the first hours of replication. Tat facilitates early transcriptional activation of proviral DNA. Rev is necessary for transport of unspliced viral mRNA to the cytoplasm. Tat and Rev are strictly required for HIV-1 replication. Nef down-regulates CD4 and major histocompatibility complex molecules, preventing presentation of viral peptides to T cells and allowing release of new virions . The structural Gag protein is the immunodominant and most conserved HIV gene product and relative or complete viral control has been associated with strong anti-Gag responses [1,2]. By inducing potent cellular immunity against Gag, Tat, Rev and Nef, one could theoretically expect the destruction of infected cells before the release of new progeny virions .
The following functional T-cell responses have been reported to be correlated with control of HIV-1 replication: preferential targeting of particular viral proteins (especially Gag) ; number or breadth of epitopes targeted [13,14]; polyfunctionality, that is simultaneous production of cytokines, such as interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and/or cytolytic factors (perforin, granzymes, CD107a expression) [15–17]; strong HIV-1-specific CD8+ T-cell proliferative responses ; and direct in-vitro CD8+ T-cell HIV-suppressive capacity .
Here we present the results of a phase I/II trial of an autologous dendritic cell-based therapeutic vaccine approach targeting Gag, Tat, Rev and Nef, optimally presented by mRNA-electroporated dendritic cells. Feasibility and safety as well as immunological and antiviral responses were assessed in patients with HIV-1 infection stable under HAART.
Materials and methods
This phase I/II study was approved by the Ethics Committee of the Antwerp University Hospital and by the Institutional Review Board of the Institute of Tropical Medicine. Following informed written consent, six adult patients with chronic HIV-1 subtype B infection and stable under HAART were included.
Dendritic cell vaccination and safety monitoring
The primary endpoint was to determine feasibility and safety of dendritic cell vaccination. Clinical-grade dendritic cell vaccines were prepared after leukapheresis of nonmobilized blood (Cobe Spectra), immunomagnetic selection of CD14+ monocytes (CliniMACS, Miltenyi Biotec) and culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 followed by maturation with TNF-α and prostaglandin E2 . Dendritic cells were then electroporated with documented-grade mRNA (generously provided by Dr Kris Thielemans, Free University of Brussels, Brussels, Belgium) encoding HIV-1 subtype B consensus Gag or a chimeric Tat-Rev-Nef protein, following in-vitro transcription of the plasmids pST1-sig-Gag-DC-LAMP (GenBank accession number AY936877) and pGEM-sig-TaReNef-DC-LAMP (GenBank accession number DQ668405), respectively [21,22], and viably cryopreserved in aliquots at −80°C. Following thawing, gag and tat-rev-nef mRNA-electroporated dendritic cells (10 × 106 each) were administered separately in a different arm, half intradermally and half subcutaneously, every 4 weeks on four occasions, designated as V1, V2, V3 and V4 (Fig. 1). Safety was evaluated by examining the patients and by drawing their blood for determination of clinical laboratory hematology and biochemistry, HIV-1 plasma viral load and absolute CD4+ and CD8+ T-cell counts.
Secondary endpoints were to assess the ability of dendritic cells to enhance HIV-specific T-cell responses. To this end, 50 ml blood was drawn 3 weeks before the first vaccination (time point T1) and 1 week after the second vaccination (T2). In addition, 500 ml blood was obtained at 1 (T3) and 6 weeks (T4) after the last vaccination. PBMCs were isolated, cryopreserved viably and thawed for later analysis.
Assessment of HIV-specific CD8+ T-cell responses
IFN-γ ELISPOT assays (Diaclone) were performed with thawed PBMCs as described elsewhere [14,23,24].
The 3H-thymidine uptake proliferation assay (Perkin Elmer) was performed on thawed PBMCs , cultured for 6 days with HIV-1 peptide pools that scored positive in IFN-γ ELISPOT.
Antigen-specific T-cell polyfunctionality
Circulating HIV-specific polyfunctional T cells were determined after restimulation with HIV-1 peptide pools by polychromatic flow cytometric analysis using a Cyflow ML flow cytometer (Partec).
Virus inhibition assay
For the virus inhibition assay (VIA) , PBMCs from the different time points were thawed, divided into two aliquots and expanded for 7 days with bi-specific CD3/CD8 or CD3/CD4 antibodies and IL-2, resulting in expansion and enrichment of the required target CD4+ and effector CD8+ T-cell populations, respectively. Separate cultures were established with CD4+ T cells infected with HIV isolates [subtype B: IIIB and BAL (NIH); subtype C: DU-174 (NIBSC); subtype D: 92UG024 (NIBSC); CRF-02: CA18 (Institute of Tropical Medicine)] at a multiplicity of infection (MOI) of 0.001. Those HIV-1-infected CD4+ target T cells were co-cultured or not with autologous CD8+ effector T cells at a ratio of 1 : 1. The percentage inhibition of viral production in CD8+/infected CD4+ cell co-cultures as compared to infected CD4+ T cells alone was calculated.
Data mining and statistical analysis
Flow cytometric data analysis was performed using FlowJo version 8.4.4 (Treestar Inc., Ashland, Oregon, USA). Graphpad Prism 4.0 (GraphPad Software Inc., La Jolla, California, USA) and SPSS 16.0 (SPSS Inc., Chicago, Illinois, USA) software were used for graphical data representations and statistical computations. Paired Student's t-test and Pearson correlation coefficient r were used to analyze normally distributed data; Wilcoxon matched pairs signed-rank test and Spearman correlation coefficient rho to examine data not normally distributed.
Feasibility and safety
The clinical details are summarized in Table 1. Vaccine production was successful in all patients from a single apheresis procedure (10–15 l). Adverse events were assessed and only local, transient and mild reactions were observed: all patients reported redness and induration at the site of injection. One patient (H022) reported fever 1 week after the first immunization. No significant alterations were observed in the hematological, biochemical and immunological parameters examined. No significant change occurred in the circulating frequencies, absolute numbers of lymphocyte subsets or plasma viral load. These data confirmed the feasibility as well as the clinical, immunological and virological safety of the vaccine.
Peripheral blood mononuclear cells from the different time points were stimulated with peptide pools containing Gag, Tat, Rev or Nef peptides in an IFN-γ ELISPOT assay (Fig. 1). When comparing post-dendritic cell with pre-dendritic cell vaccination (time points T4 vs. T1), there was an increase in magnitude of the IFN-γ ELISPOT response, that was significant for Gag (P = 0.0008, paired t-test), but not for Tat-Rev-Nef epitopes. The dendritic cell vaccine also induced increased breadth of IFN-γ response to Gag in five of six patients, that is to epitopes without a clearly positive IFN-γ response before dendritic cell vaccination (P = 0.0117, paired t-test, overall comparison of T4 vs. T1). Broadening of Tat-Rev-Nef epitopes was only observed occasionally and was not significant. When all time points were analyzed, there was a significant correlation between the magnitude and breadth of the HIV-1-specific immune response (r = 0.82, P < 0.0001). We conclude that the dendritic cells induced a concomitant increase in magnitude and breadth of IFN-γ response to HIV-1 Gag.
Next, proliferative capacity of T cells before and after immunization was evaluated (Fig. 2). There was a significant increase in stimulation index, when considering all peptide pools tested (P = 0.006, paired t-test) and Gag peptide pools (P = 0.025, paired t-test). We conclude that the dendritic cell vaccine resulted in a significant increase in proliferating T cells and that this was due, at least in part, to an anti-Gag response.
A longitudinal analysis was performed of the polyfunctional profile of HIV-1-specific CD4+ and CD8+ activated T cells, expressing IFN-γ, TNF-α, IL-2 and/or CD107a (Fig. 3) in response to short-term stimulation by Gag, Tat, Rev or Nef peptide pools. The percentage of T cells with two or more of these markers increased by at least 50% in three of six patients, without concomitant increase of monofunctional cells. When comparing T4 with T1, CD4+ T-lymphocyte polyfunctionality increased in two of six (3-marker-positive cells in H023 and H028) and CD8+ T-lymphocyte polyfunctionality in three of six patients (2-marker-positive cells in H023, 3- and 4-marker-positive cells in H025 and 2- and 3-marker-positive cells in H028). Those shifts towards more polyfunctional T cells were modest, with the exception of patient H028. There was a correlation between the percentage of polyfunctional CD4+ and CD8+ T cells, when considering all time points for T cells expressing, respectively, two markers (r = 0.68, P = 0.0003) and two or more markers (r = 0.70, P = 0.0001).
Purified CD4+ T cells were infected with the vaccine-matched HIV-1 IIIB strain and co-cultured with or without purified CD8+ T cells from the same time point. Endogenous HIV replication, evaluated by stimulating nonsuperinfected CD4+ T cells, was not detected. IIIB suppression by autologous CD8+ T cells over time is shown in Fig. 4. Before vaccination, CD8+ T cells completely lacked HIV-suppressive activity in three of five of evaluable patients (H022, H026 and H028), whereas H024 showed a limited (23.5%) suppressive capacity. In H024 and H026, suppression remained absent 1 week after the first vaccination, whereas in H022 it went up to 74% and in H028 to 100%. The virus-inhibiting capacity increased further at T3 and even more at T4 in patients H024, H026 and H028. Remarkably CD8+ T cells from patient H025 showed already 95% IIIB inhibitory activity at T1, increasing up to 100% from T2 on. There was a significant overall increase in IIIB virus inhibitory activity when comparing T4 and T1 of the five patients with available data for all four time points (P = 0.0313, Wilcoxon signed-rank test). This overall increase was not uniform for all patients: H022 showed a variable virus suppression over time.
We also evaluated the CD8+ T-cell activity against infection of CD4+ T cells with patient strains of other subtypes. Antiviral activity increased against certain strains, but the inhibition observed was not as pronounced as for IIIB and even decreased against subtype D strain 92UG024 (Fig. 5).
Correlation between immune response and HIV-suppressive capacity
Extensive statistical analysis was performed in order to determine which elements of the T-cell immune response induced by the dendritic cell vaccination [IFN-γ response magnitude and breadth, proliferation (stimulation index)] were correlated with HIV-suppressive capacity and with T-cell polyfunctionality.
Inhibition of IIIB was correlated with magnitude (r = 0.57, P = 0.032) and breadth (rho = 0.56, P = 0.036) of Gag-specific IFN-γ response at T1 and T4. Of note is that patient H022 who had the least pronounced inhibition of IIIB also displayed lack of Gag response broadening. There was no significant correlation between IFN-γ response to Tat-Rev-Nef and inhibition of IIIB.
When comparing T4 with T1, there was a significant correlation between breadth of IFN-γ response to all peptides tested and polyfunctional T cells expressing more than two markers (for both CD4+ and CD8+ T lymphocytes: rho = 0.9429, P = 0.0048); and between magnitude of IFN-γ response to Tat-Rev-Nef and CD8+ polyfunctional T cells with two or more markers (rho = 0.8857, P = 0.0333). There was a significant correlation between proliferation for all peptide pools and all time points tested and percentage of polyfunctional T cells, both for CD4+ T lymphocytes (3-marker-positive: r = 0.42, P = 0.0436; 3 or 4-marker-positive: r = 0.44, P = 0.0325) and for CD8+ T lymphocytes (2-marker-positive: r = 0.49, P = 0.0163; 2, 3 or 4-marker-positive: r = 0.44, P = 0.0316). We conclude that the cellular immune response to HIV-1 antigens is correlated with polyfunctional T-cell responses to the dendritic cell vaccine.
Immunotherapy with autologous dendritic cells electroporated with mRNA encoding Gag and a Tat-Rev-Nef fusion protein is clinically feasible and well tolerated in HIV-1 patients stable under HAART. No significant changes in plasma viral load or in CD4+ and CD8+ T-cell counts were observed.
Our dendritic cell strategy was clearly immunostimulatory, as shown by the significant increase in magnitude and breadth of HIV-1-specific IFN-γ response, in particular to Gag, and in T-cell proliferation. Breadth of IFN-γ response and T-cell proliferation were correlated with both CD4+ and CD8+ polyfunctional T-cell responses. Polyfunctional CD4+ and CD8+ T-cell responses were also correlated with each other. Importantly, dendritic cell vaccination induced or increased the capacity of CD8+ T cells to inhibit infection of autologous CD4+ T cells with the vaccine-related IIIB virus. Inhibition of some other viral strains belonging to various subtypes was also induced, but it was not always consistent. The CD8+ T-cell-mediated IIIB-inhibitory activity was correlated with magnitude and breadth of Gag-specific IFN-γ response. All these potent immunological features have been described to be associated with improved in-vivo control of HIV-1 [2,13–19]. This underscores the potential of dendritic cells in the treatment of HIV-1 infection.
IFN-γ ELISPOT and intracellular cytokine staining are reproducible assays of vaccine immunogenicity but, conceptually, are only indirect indicators of anti-HIV functions and did not predict the failure of the STEP study [27–32]. The increase of CD8+ T-cell-mediated inhibition of IIIB, a direct assay of antiviral response [19,33], was observed in all analyzable patients and adds significance to the present study. It was correlated to immune response to Gag. Our observation of the important role of the cellular immune response to Gag in controlling HIV-1 infection is reminiscent of similar findings in another setting .
Several phase I dendritic cell vaccination studies have been published, which all confirmed safety both in therapy-naive and in treated patients but with different outcomes in terms of immunogenicity and antiviral effects. A first series in treatment-naive  and in HAART-treated individuals  reported on dendritic cells loaded with, respectively, chemically and heat-inactivated autologous virus. In the treatment-naive individuals, prolonged suppression of viral load was seen in eight of 18 individuals and correlated with IL-2− and IFN-γ-expressing CD4+ T cells. In HAART-treated individuals, a significant delay of plasma viral rebound was observed after treatment discontinuation, associated with changes in HIV-1-specific CD4+ and CD8+ T-cell responses. Recently, a double-blind study in untreated patients showed that autologous dendritic cells pulsed with heat-inactivated virus resulted in a modest, but significant decrease in plasma viral load; only weak HIV-specific T-cell responses were elicited . In another study, HIV-1-infected individuals stable under HAART received dendritic cells electroporated with mRNA encoding Gag, Vpr, Rev and Nef derived from autologous pre-HAART plasma and CD40 ligand . Weak HIV-specific proliferative immune responses were induced in seven of nine patients. It is arguable whether pre-HAART plasma virus is ideal, since it represents only a limited set of variants. The proviral reservoir is known to be larger and a consensus or mosaic could induce broader and deeper responses, potentially providing a better vaccine coverage with improved capacity to prevent escape [6,36–38].
HIV-1 suppression was reported in our study (ex vivo) and in the trials using inactivated autologous virus (in vivo), but the latter approach has to deal with biohazard issues. There is still room for improvement of the antiviral effect induced by dendritic cell vaccination. In our study, T-cell polyfunctionality increase was suboptimal and CD8+ T-cell-mediated suppression of HIV-1 was not consistent for all HIV-1 isolates tested: in some patients, suppression of IIIB was variable and antiviral activity against a subtype D strain even decreased, which might imply a selective increased susceptibility to superinfection. Thus, the analysis of the incomplete nature of the antiviral immune response in some patients has led us to identify the following potential targets for future improvement: increasing antigenic spectrum and enhancing T-cell responses. Further optimization would include mRNA constructs with a broader immunogenic spectrum (e.g. archival autologous provirus or mosaic multiclade constructs) and more potent ways to enhance protective immune responses (e.g. by dendritic cells with a stronger immunostimulatory profile).
Author contributions: E.V.G., W.D.H. and N.C. performed immunological and virological assays. V.F.I.V.T. and Z.N.B. supervised the dendritic cell production in their cell therapy facility. J.W., G.V., V.F.I.V.T. and Z.N.B. supervised and analyzed experimental data. E.V., A.V.D.V., L.M., M.V. and E.F. were involved in the clinical trial design, patient selection, vaccination and clinical follow-up of the patients. E.S. and S.A. performed extensive statistical analysis and final editing of the figures. E.V.G. wrote the first draft of the manuscript. V.F.I.V.T., H.G., G.V. and Z.N.B. reviewed, edited and revised the manuscript to final version.
The work was supported by the Interuniversity Attraction Poles (IAP grant no. P6/41) of Belgian Science Policy; by grant no. 60511 of the Agency for Innovation by Science and Technology (IWT); by the Fund for Cell Therapy of the Antwerp University Hospital and by a grant of the Methusalem program of the Flemish Government to the University of Antwerp and to H.G. E.S. and N.C. hold a postdoctoral fellowship and S.A. a PhD fellowship of the Research Foundation Flanders (FWO-Vlaanderen). W.D.H. holds a PhD fellowship of the IWT. The technical assistance of Barbara Stein, Griet Nijs, Céline Merlin and Derek Atkinson is gratefully acknowledged.
Authors’ statement: All authors have read the manuscript as submitted to AIDS and have approved the content of this manuscript.
Conflicts of interest
There are no conflicts of interest.
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