Sabine D. Allard

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.

The combination of 4-1BBL and CD40L strongly enhances the capacity of dendritic cells to stimulate HIV-specific T cell responses

One of the consequences of HIV infection is a progressive loss of T cell functions, resulting in decreased cytokine secretion and proliferation and an increased sensitivity to apoptosis. Therefore, successful therapeutic vaccination approaches should aim at restoring the functionality of existing HIV‐specific T cells, as well as to efficiently induce potent, HIV‐specific T cells from naïve T cells. In this study, we wanted to determine the stimulatory capacity of DCs coelectroporated with mRNA encoding for different costimulatory molecules of the TNFSF, together with HIV antigen‐encoding mRNA. We show that DCs electroporated with 4‐1BBL can enhance the proliferation, functionality, cytokine production, and survival of HIV‐specific CD8+ T cells. Furthermore, we are the first to show that a combination of 4‐1BBL and CD40L overexpression on DCs dramatically enhances CD4+ and CD8+ T cell responses. Finally, we demonstrate that signaling through 4‐1BB, but not through CD40, can alleviate the suppressive effect of Tregs on CD8+ T cell proliferation. Thus, the combination of 4‐1BBL and CD40L enhances HIV‐specific CD8+ T cell responses in a synergistic way, resulting in enhanced proliferation of CD4+ and CD8+ T cell subsets, an increased cytokine secretion, and a reduced sensitivity to Treg‐mediated immune suppression.

Heparin-binding, Hemagglutinin-specific IFN-γ Synthesis at the Site of Infection during Active Tuberculosis in Humans

RATIONALE Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVES The mycobacterial antigen heparin-binding hemagglutinin (HBHA) induces strong IFN-γ responses by circulating lymphocytes from subjects latently infected with Mycobacterium tuberculosis, and low responses associated with CD4(+) regulatory T (Treg) cells in patients with TB. Here, we investigated HBHA-specific IFN-γ responses at the site of the TB disease. METHODS Bronchoalveolar lavages, pleural fluids, and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary or pleural TB. HBHA-specific IFN-γ production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS The percentages of HBHA-induced IFN-γ(+) alveolar and pleural lymphocytes were higher for pulmonary (P < 0.0001) and for pleural (P < 0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-γ. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSIONS Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Expansion of polyfunctional HIV-specific T cells upon stimulation with mRNA electroporated dendritic cells in the presence of immunomodulatory drugs

ABSTRACT Recently, it has been demonstrated that disease progression during HIV infection is not determined merely by the number of HIV-specific T cells but also by their quality (J. R. Almeida, et al., J. Exp. Med. 204:2473–2485, 2007; C. T. Berger, et al., J. Virol. 85:9334–9345, 2011; M. R. Betts, et al., Blood 107:4781–4789, 2006; V. V. Ganusov, et al., J. Virol. 85:10518–10528, 2011; P. Kiepiela, et al., Nat. Med. 13:46–53, 2007; and F. Pereyra, et al., J. Infect. Dis. 197:563–571, 2008). Therefore, strategies to specifically enhance or induce high-quality, HIV-specific T-cell responses are necessary to develop effective immune therapies. Thalidomide, lenalidomide, and pomalidomide have a strong capacity to boost immune responses and are therefore referred to as immunomodulatory drugs (IMiDs). We evaluated the effects of lenalidomide and pomalidomide on HIV-specific T cells. We found that the presence of IMiDs during in vitro T-cell stimulation with dendritic cells electroporated with Gag- or Nef-encoding mRNA resulted in higher numbers of cytokine-secreting HIV-specific CD8+ T cells, particularly inducing polyfunctional HIV-specific CD8+ T cells with an enhanced lytic capacity. Furthermore, CD8+ T-cell responses were detected upon stimulation with lower antigenic peptide concentrations, and a higher number of Gag epitopes was recognized upon addition of IMiDs. Finally, IMiDs reduced the proliferation of the HIV-specific CD4+ T cells while increasing the number of polyfunctional CD4+ T cells. These results provide new information about the effects of IMiDs on antigen-specific T cells and suggest that these drugs increase the efficacy of immune therapies for infectious diseases and cancer.

Lumenal part of the DC-LAMP protein is not required for induction of antigen-specific T cell responses by means of antigen-DC-LAMP messenger RNA-electroporated dendritic cells.

Previous studies showed that stimulation of T cells derived from HIV-1-infected patients with autologous dendritic cells electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses in vitro. Linking the antigen to an MHC class II-targeting sequence, such as dendritic cell lysosome-associated membrane protein (DC-LAMP), in the mRNA construct results in presentation of antigenic peptides in both MHC class I and class II molecules and therefore enhances the induced T cell responses. To analyze whether the lumenal domain of DC-LAMP is required for optimal induction of cellular immunity against HIV antigens, we compared fusion constructs with or without the lumenal domain of the DC-LAMP protein. A human codon-optimized consensus Gag sequence and a chimeric cDNA sequence encompassing Tat, Rev, and Nef codons (TaReNef ) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain. The Gag protein lacking the DC-LAMP-derived sequence altogether elicited only weak T cell responses. DCs electroporated with Gag or TaReNef linked to DC-LAMP were able to elicit similar levels of antigen-specific CD4(+) and CD8(+) T cell responses for both Gag and TaReNef, irrespective of the addition of the DC-LAMP lumenal domain. These data show that DC-LAMP-mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen-specific T cell responses.

DC immunotherapy in HIV-1 infection induces a major blood transcriptome shift

OBJECTIVE This study aimed to evaluate the effect of dendritic cell (DC) vaccination against HIV-1 on host gene expression profiles. DESIGN Longitudinal PBMC samples were collected from participants of the DC-TRN trial for immunotherapy against HIV. Microarray-assisted gene expression profiling was performed to evaluate the effects of vaccination and subsequent interruption of antiretroviral therapy on host genome expression. Data from the DC-TRN trial were compared with results from other vaccination trials. METHODS We used Affymetrix GeneChips for microarray gene expression analysis. Data were analyzed by principal component analysis and differential gene expression was assessed using linear modeling. Gene ontology enrichment and gene set analysis were used to characterize differentially expressed genes. Transcriptome analysis included comparison with PBMCs obtained from DC-vaccinated melanoma patients and of healthy individuals who received seasonal influenza vaccination. RESULTS DC-TRN immunotherapy in HIV-infected individuals resulted in a major shift in the transcriptome. Longitudinal analysis demonstrated that changes in the transcriptome sustained also during interruption of antiretroviral therapy. After DC-vaccination, the transcriptome was enriched for cellular immunity associated genes that were also induced in healthy adults who received live attenuated influenza virus vaccination. These beneficial responses were accompanied by detrimental signals of general immune activation. CONCLUSIONS The DC-TRN induced changes in the transcriptome were profound, lasting, and consisted of both protective signals and signatures of inflammation and immune exhaustion, with a net result of decreased viral load, without clinical benefit. Thus transcriptome analysis provides useful information, dissecting both positive and negative effects, for the evaluation of safety and efficacy of immunotherapeutic strategies.

HIV-1 evolution in patients undergoing immunotherapy with Tat, Rev, and Nef expressing dendritic cells followed by treatment interruption

Objectives:This study aimed to evaluate HIV sequence evolution in whole genes and in CD8+ T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. Design:HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. Methods:HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8+ T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. Results:Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8+ T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. Conclusion:Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8+ T-cell epitope level.

Sequence evolution and escape from specific immune pressure of an HIV-1 Rev epitope with extensive sequence similarity to human nucleolar protein 6

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.