Appropriate major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) responses are important for HIV-1 and simian immunodeficiency virus (SIV) disease control as evidenced by the association of virus-specific CD8+ T-cell responses and control of acute and chronic viremia [1–3]. Immunodominant CTL epitopes are those most frequently targeted during acute and chronic infection and, therefore, were the first to be identified and used as candidates for T-cell-based vaccine design . However, dominant responses frequently select escape mutations during acute infection  and targeting particular immunodominant epitopes may, therefore, be ineffective. The immunodominance of specific CD8+ T cells may, in such cases, hinder development of potentially more effective subdominant responses [6–8].
A novel vaccine approach would be to redirect the CD8+ and CD4+ T-cell immunity to target relatively ‘immunologically silent’ subdominant epitopes. A previous study has indicated the potential beneficial effects of CTLs, which target subdominant epitopes . Similar protective effects of subdominant responses are reported in the immune control of lymphocytic choriomeningitis virus (LCMV) , CMV  and pathogenic SIV . To identify subdominant and conserved CTL epitope targets, we used ‘reverse immunology’ through computerized artificial neural network predictions of HLA-A*0201 binding, followed by measurement of actual peptide binding in vitro . We tested the immunogenicity of the selected peptides in HLA-A*0201 transgenic mice and confirmed their infrequent targeting but in-vivo processing in untreated HIV-1-infected individuals [13,14]. To compensate for the high diversity of HIV-1 and investigate the potential for targeting the virus at several different highly conserved regions simultaneously , we selected seven of these novel HLA-A2 CTL epitopes derived from several proteins in HIV-1 [14,16]. Weak immunogenic target epitopes were modified into potent peptide immunogens by anchor optimization and confirmed improved immunogenicity and cross-reaction to the target epitopes [13,14]. CD4+ T-cell help is needed for induction of new CD8+ T-cell immunity. In addition, HIV-1-specific CD4+ T-cell responses are themselves associated with control of disease in infected individuals [17,18]. We, therefore, also included two HIV-1-derived epitopes and one universal non-HIV CD4+ T-cell epitope.
Dendritic cells are known to be potent stimulators of primary and secondary T-cell responses and have been used as an adjuvant for active immunotherapy in cancer patients  and HIV-infected individuals [20–23]. In this study, we used autologous monocyte-derived dendritic cells (MDDCs) generated ex vivo as carriers of the epitope immunogens. MDDCs from similar protocols have been shown to be uninfected, with normal immune functions and have served as competent antigen-presenting cells for the induction of potent T-cell responses in preliminary therapeutic HIV-1 vaccine experiments [24,25].
Here, in a test-of-concept study, we immunized treatment-naive HIV-1+, HLA-A0201+ individuals with autologous MDDCs pulsed with seven novel subdominant CTL epitopes and three CD4+ T-helper peptides to assess the possibility of inducing additional HIV-1-specific T-cell responses during chronic HIV-1 infection.
Participants and methods
The protocol was approved by the Institutional Ethical Committees of the Danish Ministry of Health (code KF02 296041) and the Danish Medicines Agency (Journal 2612-3106) and registered legally in the European Clinical Trials Database (EudraCT protocol 2006-000102-22 and ClinicalTrials.gov Protocol Registration system, NCT00856154). The study was conducted in accordance with the provisions of the Declaration of Helsinki and monitored by the Good Clinical Practice (GCP) unit at the university hospitals of Copenhagen. HIV-1+ individuals followed at university hospitals of Copenhagen and Hvidovre were included after written informed consent. Study participants had to meet the following criteria: male sex, age 18–50 years, HLA-A*0201+, HIV-1 seropositivity for 1 year or more, no clinical AIDS, CD4+ 300 cells/μl or more, never received antiretroviral therapy, hemoglobin, platelets and serum creatinine within normal range, plasma viral RNA load 1000–100 000 copies/ml, not received any other vaccine or immune-modulating medicine within 3 months and absence of other chronic, autoimmune or allergic diseases. In total, 12 participants were included in the present study with mean age 39 years (range 30–48 years), mean seropositivity 46 months (range 16–114 months), mean CD4+ T-cell counts 565 cells/μl blood (range 355–982 cells/μl blood) and geometric mean plasma viral load 8321 RNA copies/ml (range 2445–25250 RNA copies/ml). Participant 25 did not attend the visit 32 weeks after first immunization and, therefore, only the first evaluation time point was available.
Viral loads and CD4+ T-cell counts
Plasma HIV-1 RNA load was measured using the taqman real-time PCR Monitor kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. Absolute CD4+ and CD8+ T-cell counts were determined using the FACSCount system (BD Biosciences, Denmark) according to the manufacturer's protocol.
Sequencing of HIV-1
Viral RNA was extracted from plasma using QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany). Reverse transcription of RNA extract was performed essentially as described by Rousseau et al.  using primers oligo-dT and a sequence-specific primer (5′-gcactcaaggcaagctttattgaggct-3′). PCR amplification of cDNA was performed as previously described . Nested PCR amplification of four different regions covering previously described epitopes was performed using Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, California, USA) according to manufacturer's instructions, using primers 5′-ccaagaagaaaagcaaagatcattag-3′, 5′-tctagtgtccattcattgtatggctc-3′ (vif), 5′-gcatgacaaaaatcttagagccttttag-3′, 5′-tgcttgtaactcagtcttctgatttgt-3′ (pol), 5′-atctctagcagtggcgcccgaacag-3′, 5′-ggccatccattcctggctttaattttactg-3′(gag), and 5′gcagaagacagtggcaatgag-3′, 5′-caaaggatayctttggacaggc-3′ (vpu/env). Cloning of PCR products was performed using TOPO-TA cloning kit (Invitrogen). Epitope sequences were investigated by the EPILIGN software (Los Alamos database).
Synthetic peptide immunogens
Peptides were synthesized by Schafer-N (Copenhagen, Denmark). Purity of all peptides for clinical use was more than 95%. Peptides were suspended at 1 mg/ml in clinical grade Roswell Park Memorial Institute (RPMI) media, filtered through a 0.22 μm filter and tested for sterility and endotoxins. To improve immunogenicity of selected subdominant epitopes, we used primary anchor optimized variants with improved binding to HLA-A*0201 . One minimal 8mer epitope (Gag433) was not optimized, as the HLA-A*0201 binding and immunogenicity was already acceptable. The modified CD8+ T-cell epitope immunogens were Gag433 (FLGKIWPS), Gag150 (T2L, RLLNAWVKV), Vif23 (I9V, SLVKHHMYV), Env67 (V2I, NIWATHACV), Vif101 (M9L, GLADQLIHL), Vpu66 (A9V, ALVEMGHHV) and Pol606 (T9V, KLGKAGYVV) (see also Table 1).
Monocyte-derived dendritic cell preparation and immunization
Autologous MDDCs were prepared as previously described . MDDCs were divided into two pools and pulsed for 2 h at 37°C with a mixture of three (Gag433, Gag150 and Vif23) or four (Env67, Vif101, Vpu66 and Pol606) peptides, respectively. Additionally, both pools received two HIV-1-derived broad MHC class II binding peptides from Gag and gp41 [28,29] and the pan-MHC class II binding non-HIV peptide, PADRE . MDDCs were pulsed with a concentration of 40 μg/ml of each peptide, washed twice and stored at −150°C until administration. At each immunization, the participant received two subcutaneous (s.c.) injections with the two peptide/MDDC mixtures of 0.5 ml in clinical grade X-vivo-15 medium (Cambrex Corporation, East Rutherford, New Jersey, USA) each in close proximity to left and right axillary areas, respectively. The skin was pretreated with imiquimod (aldara crème) [31,32] 12 h prior to injection. We administered four immunizations weeks 0, 2, 4 and 8.
Stimulation and intracellular cytokine staining
T-cell responses were analyzed ex vivo 3 weeks prior to and 10 and 32 weeks after first immunization with peptides corresponding to the modified epitope immunogens as well as the corresponding conserved target epitopes. Pools of previously described dominant HLA-A*0201 binding peptides from HIV-1, influenza A, Epstein–Barr Virus (EBV) and CMV, CMV antigen (Aalto, Ireland) and recombinant HIV-1 p24-Gag (Aalto) were used as virus-specific controls of T-cell stimulation.
For intracellular cytokine staining (ICS), fresh whole blood was stimulated with 10 μg/ml of peptide and the costimulatory antibodies, anti-CD49d and anti-CD28 (6 μg/ml; BD Biosciences) for 6 h at 37°C with the addition of Brefeldin A (12.5 μg/ml, Sigma-Aldrich) during the last 4 h. A nonstimulated blood sample served as background control and phorbol 12-myristate 13-acetate (PMA) (2.5 μg/ml; Schafer-N) plus ionomycin (5 μg/ml; BD Biosciences) or Staphylococcus enterotoxin B (SEB) (1 μg/ml; Sigma-Aldrich, St Louis, Missouri, USA) served as positive control. Cells were subsequently fixed and permeabilized using FACS Lysing and FACS Permeabilizing solutions (BD Biosciences). Following permeabilization, cells were stained with anti-CD3 PerCP (BD Biosciences), anti-CD8 PE (Dako, Denmark) or anti-CD8 APC-Cy7 (BD Biosciences), anti-CD4 PE (Dako), anti-interferon-γ FITC (BD Biosciences), anti-interleukin-2 APC (BD Biosciences) or anti-tumor necrosis factor α Cy7PE (BD Biosciences) and subsequently fixed using 2% paraformaldehyde. Staining of CD3, CD4 and CD8 after permeabilization allowed detection of these markers even when downregulated in response to stimulation, as previously used .
Peptide-specific CD8+ T cells were detected using custom-made Pentamers (Proimmune, Oxford, United Kingdom) according to manufacturer's protocol. As controls, pentamers specific for one nonsense epitope and one cancer epitope were used. Pentamer staining was considered positive if the staining was above 2 SD of HIV-negative HLA*A02-positive individuals (n = 3).
Flow cytometry and analysis
ICS and pentamer stained cells were acquired on a BD LSRII instrument using FACSdiva software (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, Oregon, USA). Between 200 000 and 1 000 000 events in the lymphocyte gate were collected. The background level of cytokine staining in the nonstimulated sample varied, but was typically less than 0.05% of CD3+CD4+ or CD3+CD8+ lymphocytes. The samples were considered positive if the percentages of cytokine-stained cells were more than twice the background stimulation and more than 0.05% after subtracting the background level of cytokine staining in the nonstimulated sample and with a distinct population of cytokine-positive cells. A response was considered boosted if it fulfilled the above criteria and in addition, the cytokine responses were more than twice the previous evaluation time point or an additional cytokine response was induced.
Induction of CD8+ T cells specific for novel subdominant HIV-1 cytotoxic T-lymphocyte epitopes during infection
Of the 12 immunized individuals, 11 generated de-novo or boosted CD8+ T-cell responses to at least one of the CTL epitopes at 10 weeks after the first immunization, as measured by intracellular cytokine production. We stained CD8+ T-cell lymphocytes for interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) responses to both the modified epitopes and the corresponding natural target epitopes (Fig. 1). IL-2 induction was identified only for participant 28 against Pol606 at the first evaluation time point (data not shown). Single cytokine responses (primarily IFN-γ or TNF-α and rarely IL-2) were predominant in novel CD8+ T-cell responses. A relatively low frequency (0.05%) of double cytokine producing CD8+ T cells was identified in participant 2 (Pol606) (data not shown). In the case of participant 27 and participant 53, a boosted response could be identified against the CTL epitope Gag433 as indicated by a stronger IFN-γ response and in participant 53 by a novel TNF-α response, respectively. A pool of five HLA-A2 binding dominant HIV-1 epitopes and a pool of three HLA-A2 binding EBV, CMV and FLU peptides were included as virus-specific positive controls, and responses were detected in 10 and 12 participants, respectively. These responses were not altered over time and during immunization with the HIV-1 epitopes (data not shown).
We also detected epitope-specific CD8+ T cells by pentamer labeling (Fig. 2). Our screening included four different pentamers, Gag433, Pol606, Env67 and Vif23, and identified new specific CD8+ T cells in six out of 12 individuals 10 weeks after the first immunization (Fig. 2). These epitope-specific CD8+ T cells identified by pentamers correlated with intracellular IFN-γ or TNF-α responses in most but not all cases. See, for example, in participant 11, no intracellular IFN-γ or TNF-α responses could be detected, and pentamer labeling identified three additional de-novo responses.
To evaluate whether the de-novo induced CD8+ T-cell responses were sustained, we evaluated the responses 32 weeks after the first immunization. In five out of the six cases, epitope-specific CD8+ T cells first detected at 2 weeks after the last immunization were sustained at 32 weeks after the first immunization, using pentamer labeling (Fig. 2). Moreover, in participant 2 and participant 36, a lower epitope-specific CD8+ T-cell response was measured only at the second evaluation time point 32 weeks after the first immunization. Using ICS for evaluation of IFN-γ, TNF-α and IL-2, we did not find any sustained de-novo CD8+ T-cell responses (data not shown), whereas the boosted IFN-γ response against Gag433 in participant 27 remained elevated at the second evaluation time point (data not shown).
It was possible to induce new or boosted CD8+ T cells specific for at least one subdominant epitope in 12 out of 12 individuals, using ICS and/or pentamer labeling summarized in Table 1. Up to five novel induced CD8+ T-cell responses could be identified in one participant (participant 36). The most immunogenic CTL epitope was Pol606 with de-novo cytokine responses or specific CD8+ T cells in nine out of 12 participants; however, all epitopes induced novel specific CD8+ T cells in at least two participants (Table 1).
Robust CD4+ T-helper responses induced by three major histocompatibility complex class II binding peptides
We loaded MDDCs with two HIV-1-derived and one non-HIV-1-derived MHC class II binding 13-20mer peptides. Novel induced CD4+ T-cell responses were detected in at least one peptide in 10 out of 12 participants and in 11 of 12 participants 10 and 32 weeks after the first immunization, respectively (Fig. 3a and Table 1). In particular, the non-HIV peptide PADRE induced robust CD4+ T-cell responses in eight out of 12 participants. The two HIV-1-derived MHC II peptides Env570 and Gag298 each induced responses in eight participants. Novel induced helper responses were dominated by CD4+ T cells double positive for TNF-α and IL-2 production, but triple cytokine and single IFN-γ producing responses were also identified (Fig. 3a–c).
CD4+ T-cell responses present before treatment were not altered by dendritic cell immunization. Two participants (participant 53 and participant 57) exhibited CD4+ T-cell responses to the two HIV-1 peptides Gag298 and Env570 already before immunization, and the response for participant 53 was boosted to become TNF-α positive, whereas the response for participant 57 was unchanged (Fig. 3a). Moreover, CMV was included as virus-specific positive control and CD4+ T-cell responses were found in 10 of 12 participants before and after immunization (data not shown).
Effect on clinical parameters by dendritic cell immunizations with subdominant cytotoxic T-lymphocyte epitopes
Sequencing plasma HIV-1 RNA confirmed that in all individuals the majority of the epitopes or epitope variants previously shown to cross-react in HLA-A*0201 mice  were present before immunization (Table 1). We followed plasma HIV-1 viral load and CD4+ T-cell counts for 1 year prior to and up to 8 months after the dendritic cell immunization (Fig. 4). Initial transient decreased viral loads were observed in five participants (participants 13, 25, 37, 42 and 57). The most pronounced changes were seen in participant 13, who exhibited a 1.08 (4.26–3.18) log10 viral load reduction starting after the first immunization and lasting up to 2 weeks after the last dendritic cell immunization. The CD4+ T-cell counts in this participant were stable but low during the 10 weeks immunization schedule. However, his vaccine induced drop in viral load had rebounded to pre-vaccination levels 163 days after the first immunization, which together with a stable but low CD4 count resulted in HAART initiation. We did not see any overall or sustained change in viral load after immunization (geometric mean log10 3.97 day 0 and log10 3.82 day 58). The CD4+ T-cell counts gradually decreased from mean of 482 cells/μl at day 0 to 434 cells/μl at day 252 (10%), in line with CD4+ T-cell decline over the same period in treatment-naïve individuals.
The immunizations were safe and well tolerated. No allergic or autoimmune reactions were observed. No hematological, hepatic, muscular, pulmonary or renal toxicities were observed by blood testing (data not shown). The most common reaction was mild-to-moderate local irritation at the site of subsequent injections but not at the initial injection. Interestingly, induction of CD4+ T-cell responses did not cause a raise in viral load.
This test-of-concept study is the first to evaluate the safety and immunogenicity of selected subdominant CD8+ T-cell epitopes in treatment-naïve HIV-1-infected individuals. We hypothesized that delivering multiple subdominant CD8+ T-cell epitopes in combination with HIV-1 CD4+ T-helper peptides on potent MDDCs would have the ability to induce new and functional anti-HIV-1 CD8+ T-cell responses in vivo. Using anchor-optimized epitopes, binding to HLA-A0201 and immunogenicity was improved . Targeting conserved epitopes, not targeted during natural infection, may have the potential to reduce plasma viral load and ultimately postpone the onset of HAART or AIDS, as a novel therapeutic strategy.
Immunization induced novel epitope-specific CD8+ T-cell and/or CD4+ T cells in 12 out of 12 individuals (measured by ICS and/or pentamer labeling). Thus, as a mode of immunization, autologous MDDCs pulsed with peptides, was successful even in the presence of the immune response induced by viral antigen load during chronic HIV-1 infection. Additionally, multiple novel epitope-specific CD8+ and CD4+ T cells could be generated in each participant. All seven CTL peptides and all three T-helper peptides induced novel epitope specific responses in at least two participants.
The natural CTL responses against HIV-1 are known to be important in the control of infection [1–3]. We, therefore, believe that a successful therapeutic vaccination should not reduce or alter already existing CTL responses, but merely add selected specific T-cell responses, hopefully contributing to improved control of the disease. In our study, inducing novel CD8+ and CD4+ T cells responses did not appear to alter the already existing T-cell responses toward dominant CTL or T-helper epitopes targeting HIV-1, CMV, EBV or Flu. Only in two participants responses toward subdominant epitopes already recognized before dendritic cell immunization were boosted. This might be due to the continuously high antigen exposure driving CD8+ T cells to an exhausted state not capable of responding to booster immunizations [34–37]. Exhausted antigen-specific CD8+ T cells lacking functional cytokine responses  may also explain some of the discrepancy between the antigen-specific CD8+ T cells as detected by pentamers versus cytokine production. Indeed, we found that the majority of vaccine-induced IFN-γ and TNF-α producing CD8+ T cells co-expressed high levels of CD57 (data not shown), supporting the theory of exhausted a response with a potential limited life span. Due to material limitations, this labeling was only preformed on a limited number of samples.
The majority of the induced CD4+ T-cell responses were sustained at the last evaluation time point, 32 weeks after the first immunization, whereas the novel CD8+ T-cell responses were rarely persistent. These findings may be explained by the cytokine profile of the induced responses. CD4+ T-cell responses were dominated by TNF-α and IL-2 double positive responses and some triple positive TNF-α, IL-2 and IFN-γ responses, indicative of long-lived central memory cells . In the case of CD8+ T cells, the dominating responses were single cytokine, IFN-γ or TNF-α, producing cells and no IL-2 production, indicative of short-lived effector memory cells or terminal effectors . Induction of stronger CD4+ T-cell responses compared with CD8+ T-cell responses was similarly observed in a recent study of therapeutic vaccination of chronically SIVmac251-infected pigtail macaques, using peripheral blood mononuclear cell (PBMC) pulsed with inactivated SIV .
The immunization seemed associated with a reduction of viral load in half of the immunized participants already within the first 2 weeks after the first dendritic cell immunization. This limited and transient decrease in viral load may be a sign of insufficient boosting by the cell-based immunization and/or driven by the HIV-1 infection. It is possible that the first immunization generated CTL that targeted the subsequently injected peptide-loaded MDDCs and thereby prevented (or limited) any booster effect. The increasing local reactions observed only after subsequent autologous MDDC immunizations but not after the first could support this finding. Another possibility is that the virus generates escape mutations and thereby avoids the novel induced CTLs . The affect on viral load was most pronounced in participant 13. However, plasma viral load rebounded about 3 months later, resulting in HAART initiation. The fact that this rebound in viral load appeared a relatively long time after the last immunization prompts us to believe that this was not caused by the immunizations.
Although the selected epitopes used in this study were identified within conserved regions of the HIV-1 genome, we do not know the importance of any associated mutations on viral fitness. This strategy of therapeutic vaccination could be further improved by the use of epitopes with associated escape mutations known to have an impact on viral fitness, possibly also restricted to other more relevant HLA types .
We have shown that it is possible to generate new T-cell responses against selected subdominant, but conserved, epitopes in treatment-naive HIV-1-infected individuals despite ongoing HIV-1 antigenic exposure from their chronic HIV-1 infection. This ability to redirect the immune response toward selected relatively immune-silent epitopes is an important proof of concept. Further optimization of immunization strategies could potentially lead to stronger and more durable cellular responses to additional epitopes with the potential to control viral replication better during chronic and/or acute HIV-1 infection. A reduction of viral load would protect individuals against disease progression (prevention of disease) and supplement or avoid the need for harmful and costly antiretroviral drugs. An effective T-cell-based therapeutic vaccine, by lowering viral load, would also minimize the risk of transmission to healthy individuals and thereby provide a prophylactic effect with large impact in high endemic areas.
We are grateful for the expert opinion and the helpful comments and discussions by Philip Goulder and Gregers Gram and critical review by Rebecca Payne. We acknowledge the technical assistance of Birgit Knudsen, Solvej Jensen, Irene Jensen, Anne Jensen, Kirsten Bødker, Phillippa Collins, Dorthe Petersen, Eva Gaardsdal, Charlotte Vajhøj and Heidi Bonde Knudsen. This work was supported by grants from the Danish AIDS Foundation.
Author contributions: A.F. designed research; H.N.K., J.B., M.T., L.V., A.E.P., J.L.H., B.S.A., I.M.S., J.G. and G.K. performed research; H.N.K., I.K. and L.V. analyzed data; H.N.K., I.K. and A.F. wrote the paper.
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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
cytotoxic T-lymphocyte epitopes; dendritic cells; HIV-1; subdominant epitopes; therapeutic vaccine