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Immunization of HIV-1-Infected Persons With Autologous Dendritic Cells Transfected With mRNA Encoding HIV-1 Gag and Nef: Results of a Randomized, Placebo-Controlled Clinical Trial.
Feb. 2016 | Gandhi, Rajesh T; Kwon, Douglas S; Macklin, Eric A; Shopis, Janet R; McLean, Anna P; McBrine, Nicole; Flynn, Theresa; Peter, Lauren; Sbrolla, Amy; Kaufmann, Daniel E; Porichis, Filippos; Walker, Bruce D; Bhardwaj, Nina; Barouch, Dan H; Kavanagh, Daniel G
HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy. PMID 26379068

HIV immunotherapy comes of age: implications for prevention, treatment and cure.
Feb. 2016 | Routy, Jean-Pierre; Mehraj, Vikram; Cao, Wei
Antiretroviral therapy (ART) has reshaped the lives of millions of individuals infected with human immunodeficiency virus (HIV). Patients initiating ART early in the course of infection benefit from a considerable reduction in the risks of acquired immune deficiency syndrome (AIDS) and HIV-related inflammatory events. However, the absence of cure and lifelong requirements of treatment highlight the need of a vaccine and an immunotherapeutic strategy. Like for cancer, a paradigm shift has occurred with the contribution of immune activation and microbial translocation priming aberrant systemic immunity in restricting the ability of the host to mount an effective immune response. The approaches of implementing an effective vaccine to prevent infection and inhibition of immune activation with breakage of viral latency followed by vaccination should lead to an HIV-free generation. PMID 26629806

Therapeutic Vaccination With Dendritic Cells Loaded With Autologous HIV Type 1-Infected Apoptotic Cells.
Jan. 2016 | Macatangay, Bernard J C; Riddler, Sharon A; Wheeler, Nicole D; Spindler, Jonathan; Lawani, Mariam; Hong, Feiyu; Buffo, Mary J; Whiteside, Theresa L; Kearney, Mary F; Mellors, John W; Rinaldo, Charles R
 We report the results of a phase I/II, open-label, single-arm clinical trial to evaluate the safety and anti-human immunodeficiency virus type 1 (HIV-1) efficacy of an autologous dendritic cell (DC)-based HIV-1 vaccine loaded with autologous HIV-1-infected apoptotic cells. PMID 26647281

Decreased HIV-specific T-regulatory responses are associated with effective DC-vaccine induced immunity.
März 2015 | Brezar, Vedran; Ruffin, Nicolas; Richert, Laura; Surenaud, Mathieu; Lacabaratz, Christine; Palucka, Karolina; Thiébaut, Rodolphe; Banchereau, Jacques; Levy, Yves; Seddiki, Nabila
The role of regulatory T cells (Tregs) in vaccination has been poorly investigated. We have reported that vaccination with ex vivo-generated dendritic-cells (DC) loaded with HIV-lipopeptides (LIPO-5-DC vaccine) in HIV-infected patients was well tolerated and highly immunogenic. These responses and their relation to viral replication following analytical treatment interruption (ATI) were variable. Here, we investigated whether the presence of HIV-specific Tregs might explain these differences. Co-expression of CD25, CD134, CD39 and FoxP3 was used to delineate both antigen-specific Tregs and effectors T cells (Teffs). Median LIPO-5 specific-CD25+CD134+ polyfunctional T cells increased from 0.1% (IQR 0-0.3) before vaccination (week -4) to 2.1% (IQR 1.1-3.9) at week 16 following 4 immunizations (p=0.001) and were inversely correlated with maximum viral load following ATI (r=-0.77, p=0.001). Vaccinees who displayed lower levels of HIV-specific CD4+CD134+CD25+CD39+FoxP3+ Tregs responded better to the LIPO-5-DC vaccine. After vaccination, the frequency of HIV-specific Tregs decreased (from 69.3 at week -4 to 31.7% at week 16) and inversely correlated with HIV-specific IFN-γ-producing cells (r=-0.64, p=0.002). We show that therapeutic immunization skewed the HIV-specific response from regulatory to effector phenotype which impacts on the magnitude of viral replication following ATI. PMID 25816350

Challenges in dendritic cells-based therapeutic vaccination in HIV-1 infection Workshop in dendritic cell-based vaccine clinical trials in HIV-1.
Aug. 2011 | García, Felipe; Routy, Jean-Pierre
Therapeutic immunization has been proposed as an approach that might help limit the need for lifelong combined antiretroviral therapy (cART). One approach for therapeutic vaccination which has been explored during the last few years is the administration of autologous monocyte-derived DCs (MD-DCs) loaded ex vivo with a variety of antigens. It has been shown in experimental murine models as well as in cancer patients and in patients with chronic infections that this approach can induce and potentiate antigen-specific T-cell response (and to induce a potent protective immunity). Contrary to the wide experience with this strategy in cancer, in HIV-1 infection the experience is limited and the design of the clinical trials varies greatly between groups. This variability affects all the steps of the process, from preparation of immunogen and DCs to clinical trial design and immune monitoring. Although both the study designs and the DC preparation (the maturation stimuli and the identity and source of HIV-1 antigens used to pulse DCs) varied in most of the studies that were published so far, overall the results indicate that DC immunotherapy elicits some degree of immunological response. To address this situation and to allow comparison between trials a panel of experts working in DC-based clinical trials in HIV-1 infection met in Barcelona at the end of 2010. During this meeting, the participants shared the data of their current research activities in this field in order to unify criteria for the future. This report summarizes the present situation of the field and the discussions and conclusions of this meeting. PMID 21791232

Monocyte-derived dendritic cells from HIV type 1-infected individuals show reduced ability to stimulate T cells and have altered production of interleukin (IL)-12 and IL-10.
Mai 2009 | Buisson, Sandrine; Benlahrech, Adel; Gazzard, Brian; Gotch, Frances; Kelleher, Peter; Patterson, Steven
Monocyte-derived dendritic cells (MDDCs) have been used in therapeutic vaccination for cancer. A small number of studies have employed a similar approach to vaccinate human immunodeficiency virus (HIV)-infected individuals. We have thus analyzed the functional properties of MDDCs generated from HIV-infected individuals who either are receiving highly active antiretroviral therapy or are therapy naive. There was no difference in the MDDC phenotype or efficiency of MDDC generation between HIV-infected individuals and healthy control subjects. Despite this, the MDDCs derived from both groups of infected individuals were severely impaired in their ability to stimulate the proliferation of allogeneic T cells. Furthermore, production of interferon-gamma was reduced in T cells stimulated by MDDCs. These functional changes may be at least partly explained by reduced interleukin-12 and increased interleukin-10 secretion on stimulation with lipopolysaccharide and CD40 ligand. Our findings suggest that MDDCs used in therapeutic vaccination of HIV-infected individuals may show reduced potency. PMID 19419334

Production of a dendritic cell-based vaccine containing inactivated autologous virus for therapy of patients with chronic human immunodeficiency virus type 1 infection.
Feb. 2009 | Whiteside, Theresa L; Piazza, Paolo; Reiter, Amanda; Stanson, Joanna; Connolly, Nancy C; Rinaldo, Charles R; Riddler, Sharon A
In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8(+) and CD4(+) T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4(+) T cells with the virus; (iii) inactivation of the virus in CD4(+) T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4(+) T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4(+) T cells. CD4(+) T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID(50); which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4(+) T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID(50) of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 microg/ml) and UVB irradiation (312 nm) reduced the TCID(50) of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4(+) T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137). PMID 19038780

In vitro priming recapitulates in vivo HIV-1 specific T cell responses, revealing rapid loss of virus reactive CD4 T cells in acute HIV-1 infection.
Jan. 2009 | Lubong Sabado, Rachel; Kavanagh, Daniel G; Kaufmann, Daniel E; Fru, Karlhans; Babcock, Ethan; Rosenberg, Eric; Walker, Bruce; Lifson, Jeffrey; Bhardwaj, Nina; Larsson, Marie
The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission. PMID 19165342

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef.
Juni 2008 | Allard, Sabine D; Pletinckx, Katrien; Breckpot, Karine; Heirman, Carlo; Bonehill, Aude; Michiels, Annelies; van Baalen, Carel A; Gruters, Rob A; Osterhaus, Albert D M E; Lacor, Patrick; Thielemans, Kris; Aerts, Joeri L
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. PMID 18539368

Induction of HIV-specific T and B cell responses with a replicating and conditionally infectious lentiviral vaccine.
Apr. 2008 | Wingard, Jeremy B; Anderson, Bart; Weissman, Drew
The development of an HIV vaccine that induces broad and potent immunity is critically needed. Viruses, including lentiviruses, have been used as vectors for ex vivo transduction of antigens into dendritic cells (DC). We hypothesized that DC transduced with a vector that allows selective infection of DC could induce potent immunity by continually priming DC. A lentiviral vector encoding HIV gag-pol without env would form viral cores in transduced DC, but would release non-infectious particles by budding into endosomes and releasing apoptotic bodies or exosomes containing viral cores. DC function by endocytosing DC-derived apoptotic bodies, and they are specialized in their ability to move endocytic contents into the cytoplasm. We postulated that endocytosis of vector cores could lead to transduction of a second round of DC. In this report, we demonstrate accumulation of viral cores inside transduced DC and show second-round transduction of immature DC that endocytose transduced DC in vitro. The effectiveness of immunization of mice with transduced DC to induce specific lymphocyte activation was assessed. Mice developed antigen-specific T cell responses and specific antibodies after immunization. Transduction of DC with a replication-competent but conditionally infectious lentivirus could be a novel vaccine strategy for HIV. PMID 18412164

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.
Apr. 2008 | Nchinda, Godwin; Kuroiwa, Janelle; Oks, Margarita; Trumpfheller, Christine; Park, Chae Gyu; Huang, Yaoxing; Hannaman, Drew; Schlesinger, Sarah J; Mizenina, Olga; Nussenzweig, Michel C; Uberla, Klaus; Steinman, Ralph M
DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs. PMID 18324335

Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe and induces immunogenicity in HIV-1-infected individuals.
Feb. 2008 | Connolly, Nancy C; Whiteside, Theresa L; Wilson, Cara; Kondragunta, Venkatswarlu; Rinaldo, Charles R; Riddler, Sharon A
Treatments for human immunodeficiency virus type 1 (HIV-1)-positive individuals that augment HIV-1 suppression and have potential for achieving long-term control of HIV-1 viremia in the absence of antiretroviral therapy (ART) are urgently needed. We therefore conducted a phase I, clinical safety trial of a dendritic cell (DC)-based vaccination strategy as immunotherapy for HIV-1-positive individuals on ART. We studied 18 HIV-1-positive subjects on ART who underwent leukapheresis to obtain peripheral blood mononuclear cells for DC generation from monocytes cultured with cytokines. Mature DC were pulsed with three HIV-1 HLA*A0201 Gag, Env, and Pol peptides and one influenza A virus matrix protein peptide. The vaccine was administered to donors randomized to receive two vaccinations, either intravenously or subcutaneously. The primary end points were safety and tolerability of two doses of peptide-DC vaccine (3 million versus 10 million). Secondary end points included gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses and clinical correlates of an immune response to vaccination. Autologous DC-peptide vaccine was safe, well tolerated, and feasible for use in all participants. Adverse events were rare. Although the trial was not powered to assess an immunologic response, a significantly increased frequency of HIV-1 peptide-specific IFN-gamma-positive cells was observed 2 weeks following the second vaccine, with three individuals responding to all four peptides. DC vaccination was safe, was feasible, and showed promise of immunogenicity in ART-treated, HIV-1-positive individuals. Additional studies of DC immunization strategies for HIV-1 infection are warranted. PMID 17942609

Antigen crosspresentation by human plasmacytoid dendritic cells.
Sep. 2007 | Hoeffel, Guillaume; Ripoche, Anne-Claire; Matheoud, Diana; Nascimbeni, Michelina; Escriou, Nicolas; Lebon, Pierre; Heshmati, Farhad; Guillet, Jean-Gérard; Gannagé, Monique; Caillat-Zucman, Sophie; Casartelli, Nicoletta; Schwartz, Olivier; De la Salle, Henri; Hanau, Daniel; Hosmalin, Anne; Marañón, Concepción
Crosspresentation is a specialized function of myeloid dendritic cells (mDCs), allowing them to induce CD8+ T cell responses against exogenous antigens that are not directly produced in their cytotosol. Human plasmacytoid DCs (pDCs) are not considered so far as able to perform crosspresentation. We showed here that purified human pDCs crosspresented vaccinal lipopeptides and HIV-1 antigens from apoptotic cells to specific CD8+ T lymphocytes. Apoptotic debris were internalized by phagocytosis and the lipopeptide LPPol reached nonacidic endosomes. This crosspresentation was amplified upon influenza virus infection. Importantly, the efficiency of crosspresentation by pDCs was comparable to that of mDCs. This property of human pDCs needs to be taken into account to understand the pathogenesis of infectious, allergic, or autimmune diseases and to help achieve desired responses during vaccination by targeting specifically either type of DCs. PMID 17869134

Levels of antigen processing machinery components in dendritic cells generated for vaccination of HIV-1+ subjects.
Aug. 2007 | Connolly, Nancy; Riddler, Sharon; Stanson, Joanna; Gooding, William; Rinaldo, Charles R; Ferrone, Soldano; Whiteside, Theresa L
To evaluate expression of the antigen processing machinery (APM) components and HLA molecules by monocyte-derived dendritic cells (DC) generated from chronically HIV-1 infected subjects on antiretroviral therapy (ART) and to assess their ability to ex vivo induce HIV-1 specific T cells. PMID 17690565

Robust CD4+ and CD8+ T cell responses to SIV using mRNA-transfected DC expressing autologous viral Ag.
Aug. 2007 | Melhem, Nada M; Liu, Xiang Dong; Boczkowski, David; Gilboa, Eli; Barratt-Boyes, Simon M
A potentially powerful strategy for therapeutic HIV vaccination is the use of DC transfected with mRNA encoding autologous viral Ag, as epitopes presented by transfected DC would exactly reflect those expressed by infected cells in the individual. Using human and rhesus macaque monocyte-derived DC, we show that nucleofection is a superior method for mRNA transfection, resulting in high-level protein expression and DC maturation. DC transfected with SIV gag isolated from an infected monkey stimulated robust Ag-specific recall T cell responses of similar magnitude to those induced by peptide-pulsed PBMC that were predominantly CD8+ T cell mediated. Enhanced CD4+ T cell responses were stimulated when Gag was redirected into the lysosomal pathway via the targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1). Rhesus DC transfected with lysosome-targeted gag encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region. Finally, gag-transfected DC from SIV-infected monkeys stimulated significant Ag-specific recall T cell responses in an entirely autologous system. These findings demonstrate that mRNA-transfected DC expressing SIV Ag derived from infected monkeys stimulate broad and relevant T cell responses, supporting this approach for therapeutic HIV vaccine development. PMID 17615585

Dendritic cell-based vaccine strategy against human immunodeficiency virus clade C: skewing the immune response toward a helper T cell type 2 profile.
Apr. 2007 | Gruber, Andreas; Chalmers, Alistair S; Rasmussen, Robert A; Ong, Helena; Popov, Sergei; Andersen, Janet; Hu, Shiu-Lok; Ruprecht, Ruth M
Given the continued spread of human immunodeficiency virus (HIV)-1 worldwide, developing efficient vaccine strategies against HIV-1 is a key task. We tested the safety and immunogenicity of a multicomponent, cell-based vaccine that consisted of antigen-expressing apoptotic bodies with or without autologous dendritic cells (DCs). The vaccine strategy involved transfection of human 293T cells with codon-optimized DNA vectors expressing env of HIV1084i, a newly transmitted pediatric HIV-1 clade C strain; SHIV89.6P tat; and SIVmac239 gag-protease. Apoptotic bodies were generated by heat shock and ultraviolet irradiation and mixed either with mouse DCs (DC-cell vaccine) or given directly (cell-only vaccine) to BALB/c mice for initial priming; boosts consisted of apoptotic bodies only. The immunogens were well tolerated with or without DCs. Compared with the cell-only vaccine, the DC-cell vaccine induced higher antibody titers against all three antigens, whereas virus-specific cytotoxic T lymphocyte responses were equally strong in both groups. Iso-type analysis of viral antigen-specific antibodies revealed a skewing toward helper T type 2 responses induced by the DC-cell vaccine but not by the cell-only vaccine. In summary, both vaccine strategies were safe and induced cellular as well as humoral antiviral immunity; the DC-based approach had the advantage of significantly stronger antibody responses. PMID 17425430

Understanding and exploiting dendritic cells in human immunodeficiency virus infection using the nonhuman primate model.
März 2007 | Barratt-Boyes, Simon M; Brown, Kevin N; Melhem, Nada; Soloff, Adam C; Gleason, Sherrianne M
Dendritic cells (DC) are pivotal cells in the innate immune system. Recent interest in the role of DC in human immunodeficiency virus (HIV) pathogenesis has increased with the finding that both myeloid (mDC) and plasmacytoid DC (pDC) are lost from blood during infection, associated with progression to disease. DC are also being studied intensively for their capacity to stimulate robust virus-specific immunity as vaccines. Here we discuss our work in these contrasting fields of DC biology using the rhesus macaque nonhuman primate model. We focus on studies of DC dynamics in lymphoid tissues during pathogenic simian immunodeficiency virus (SIV) infection, DC trafficking in health and disease, DC-based vaccination and the use of autologous virus as antigen for stimulation of virus-specific T cells. PMID 17337787

A dendritic cell-based vaccine for treating HIV infection: background and preliminary results.
Jan. 2007 | Andrieu, J M; Lu, W
Antibody response against human immunodeficiency virus-1 (HIV) is ineffective and cellular immune response is not strong enough to achieve the complete suppression or at least a strong control of viral replication in HIV- infected patients. In 2001, we showed in vitro that dendritic cells (DCs) of HIV-infected patients loaded with autologous HIV chemically inactivated by aldrithiol-2 were capable of raising an HIV-specific cellular immune response powerful enough to allow the destruction of autologous HIV- infected CD4 T cells. In 2003, we showed that simian immunodeficiency virus (SIV)-infected macaques vaccinated with inactivated SIV-loaded autologous DCs raised a strong SIV-specific cellular response. Ten months after vaccination, plasma viral load of 7 out of the 10 vaccinated monkeys remained 1000-fold lower than initially. In December 2004, we published results observed in 18 untreated HIV-infected patients vaccinated with autologous monocyte-derived DCs loaded with autologous inactivated HIV. A year following vaccination, 8 patients had a plasma viral load decrease >90%; among them, 4 had viral load <1000 copies mL(-1). Moreover, by one year, the viral load decline of the 18 patients was significantly correlated with their percentage of HIV-1-gag-specific CD8(+) T cells expressing perforin and that of HIV-1-specific CD4(+) T(H)1 cells. This is the first demonstration of the capacity of a therapeutic vaccine to induce an effective HIV-specific T cell response associated with sustained viral suppression in untreated viremic patients. The manipulation of antigen presenting cells to elicit virus-specific cellular responses is a promising tool to control persistant viral infections. PMID 17241177

Peptide-loaded dendritic-cell vaccination followed by treatment interruption for chronic HIV-1 infection: a phase 1 trial.
Apr. 2006 | Ide, Fuyuaki; Nakamura, Tetsuya; Tomizawa, Mariko; Kawana-Tachikawa, Ai; Odawara, Takashi; Hosoya, Noriaki; Iwamoto, Aikichi
Immune response enhanced by therapeutic HIV-1 vaccine may control viral proliferation after discontinuation of highly active antiretroviral therapy (HAART). Although which strategies for therapeutic vaccination are feasible remains controversial, application of dendritic cells (DCs) as a vaccine adjuvant represents a promising approach to improving deteriorated immune function in HIV-1-infected individuals. The safety and efficacy of DC-based vaccine loaded with HIV-1-derived cytotoxic T lymphocytes (CTL) peptides were thus investigated in this study. Autologous DCs loaded with seven CTL peptides with HLA-A*2402 restriction were immunized to four HIV-1-infected individuals under HAART. In terms of safety, peptide-loaded DCs were well tolerated, and only mild local and general symptoms were observed during vaccine administration. ELISPOT assays to detect IFN-gamma production in CD8(+) lymphocytes revealed a limited breadth of responses to immunized peptides in two of four participants, but no response in the remaining two participants. Differences in immunological response might be attributable to the fact that responders displayed higher nadir CD4 counts before starting HAART and were immunized with a larger number of DCs per reactive peptide than non-responders. Discontinuation of HAART after vaccination failed to lower viral set points compared to those before starting HAART. This early outcome warrants further exploration to elucidate the therapeutic value of vaccination with DCs in HIV-1 infection. PMID 16628588

Robust HIV-specific immune responses were induced by DNA vaccine prime followed by attenuated recombinant vaccinia virus (LC16m8 strain) boost.
März 2006 | Shinoda, Kaori; Xin, Ke-Qin; Kojima, Yoshitsugu; Saha, Sukumar; Okuda, Kentaro; Okuda, Kenji
Recombinant vaccinia virus-based vaccine combined with DNA vaccine has produced a protective immune response against HIV infection in non-human primates. In this study, we explored the immunogenicity of a recombinant vaccinia virus (LC16m8 strain), which has been used in children without severe side effects. The vaccinia virus expressing an HIV(89.6)env gene (vLC-Env) alone or combined with a DNA vaccine expressing the HIV(89.6)env gene (pCAG-Env) was characterized in BALB/c mice. Vaccination of vLC-Env induced much higher HIV-specific humoral and cell-mediated immune responses than that of pCAG-Env. Priming with pCAG-Env further enhanced vLC-Env induced immune responses, especially cell-mediated immune response. Moreover, efficient expression of Env protein was achieved following infection of bone marrow dendritic cells by vLC-Env in vitro. Administration of vLC-Env-infected dendritic cells to mice generated a high cell-mediated immune response. These results demonstrate that priming with pCAG-Env and boosting with vLC-Env represents a logical candidate for vaccination against HIV infection. PMID 16458074

Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins.
Feb. 2006 | Van Gulck, Ellen R A; Ponsaerts, Peter; Heyndrickx, Leo; Vereecken, Katleen; Moerman, Filip; De Roo, Ann; Colebunders, Robert; Van den Bosch, Glenn; Van Bockstaele, Dirk R; Van Tendeloo, Viggo F I; Allard, Sabine; Verrier, Bernard; Marañón, Concepción; Hoeffel, Guillaume; Hosmalin, Anne; Berneman, Zwi N; Vanham, Guido
Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigen-loaded dendritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. In the present study, monocyte-derived DCs from nontreated HIV-1-seropositive patients were electroporated with codon-optimized ("humanized") mRNA encoding consensus HxB-2 (hHXB-2) Gag protein. These DCs elicited a strong HIV-1 Gag-specific interferon-gamma (IFN-gamma) response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DCs also triggered IFN-gamma secretion by autologous peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD8+ T cells from all patients tested. Next, a novel strategy was developed using autologous virus sequences. Significant specific IFN-gamma T-cell responses were induced in all patients tested by DCs electroporated with patients' autologous polymerase chain reaction (PCR)-amplified and in vitro-transcribed proviral and plasma viral mRNA encoding either Gag or Env. The stimulatory effect was seen on PBMCs, CD8+ T cells, and CD4+ T cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin-2 (IL-2) T-cell response was induced by DCs electroporated with hHxB-2 or proviral gag mRNA. These findings open a major perspective for the development of patient-specific immunotherapy for HIV-1 disease. PMID 16263796

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef.
Feb. 2006 | Kavanagh, Daniel G; Kaufmann, Daniel E; Sunderji, Sherzana; Frahm, Nicole; Le Gall, Sylvie; Boczkowski, David; Rosenberg, Eric S; Stone, David R; Johnston, Mary N; Wagner, Bradford S; Zaman, Mohammad T; Brander, Christian; Gilboa, Eli; Walker, Bruce D; Bhardwaj, Nina
Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy. PMID 16249391

Long-term HIV-specific responses and delayed resumption of antiretroviral therapy after peptide immunization targeting dendritic cells.
Feb. 2006 | Kran, Anne-Marte B; Sørensen, Birger; Sommerfelt, Maja A; Nyhus, Jørgen; Baksaas, Ingebjørg; Kvale, Dag
Long-term HIV-specific immune responses and clinical outcomes were evaluated in HIV-infected patients previously immunized with p24-like peptides (Vacc-4x) targeting dendritic cells (DC). Vacc-4x-induced cellular immune responses were unchanged 1.5 years after completing immunization, and 62% were still off combined antiretroviral treatment (CART). The magnitude of early Vacc-4x responses determined whether the resumption of CART was clinically indicated 2 years after enrollment. These observations encourage further exploration of both Vacc-4x and other HIV peptide-based immunization regimens targeting DC. PMID 16470131

Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection.
Jan. 2006 | Wiley, Rebecca D; Gummuluru, Suryaram
Immature dendritic cells (DCs) capture HIV type 1 (HIV-1) and can transmit captured virus particles to T cells. In this report, we show that HIV-1 particles captured by DCs can be transmitted to T cells by exocytosis without de novo infection. Captured HIV-1 particles were rapidly endocytosed to tetraspan protein (CD9, CD63)-positive endocytic compartments that were reminiscent of multivesicular endosomal bodies. Furthermore, some of the endocytosed virus particles were constitutively released into the extracellular milieu in association with HLA-DR1(+), CD1b(+), CD9(+), and CD63(+) vesicles (exosomes) and could initiate productive infections of CD4(+) target cells. Surprisingly, the exocytosed vesicle-associated HIV-1 particles from DCs were 10-fold more infectious on a perparticle basis than cell-free virus particles. These studies describe a previously undescribed mechanism of DC-mediated HIV-1 transmission and suggest that virus particle trafficking to multivesicular endosomal bodies and subsequent exocytosis can provide HIV-1 particles captured by DCs an avenue for immune escape. PMID 16407131

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy.
Jan. 2006 | Dullaers, Melissa; Thielemans, Kris
Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC. This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented. PMID 16288497

Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions.
Nov. 2005 | Beignon, Anne-Sophie; McKenna, Kelli; Skoberne, Mojca; Manches, Olivier; DaSilva, Ida; Kavanagh, Daniel G; Larsson, Marie; Gorelick, Robert J; Lifson, Jeffrey D; Bhardwaj, Nina
HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS. PMID 16224540

Processing and presentation of exogenous HLA class I peptides by dendritic cells from human immunodeficiency virus type 1-infected persons.
Feb. 2005 | Huang, Xiao-Li; Fan, Zheng; Colleton, Bonnie A; Buchli, Rico; Li, Hongyi; Hildebrand, William H; Rinaldo, Charles R
Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8(+) T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8(+) T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection. PMID 15709025

Therapeutic dendritic-cell vaccine for chronic HIV-1 infection.
Dez. 2004 | Lu, Wei; Arraes, Luiz Claudio; Ferreira, Wylla Tatiana; Andrieu, Jean-Marie
We present the results of a preliminary investigation of the efficacy of a therapeutic dendritic cell (DC)-based vaccine for HIV-1. We immunized 18 chronically HIV-1-infected and currently untreated individuals showing stable viral loads for at least 6 months with autologous monocyte-derived DCs loaded with autologous aldrithiol-2-inactivated HIV-1. Plasma viral load levels were decreased by 80% (median) over the first 112 d following immunization. Prolonged suppression of viral load of more than 90% was seen in 8 individuals for at least 1 year. The suppression of viral load was positively correlated with HIV-1-specific interleukin-2 or interferon-gamma-expressing CD4(+) T cells and with HIV-1 gag-specific perforin-expressing CD8(+) effector cells, suggesting that a robust virus-specific CD4(+) T-helper type 1 (T(H)1) response is required for inducing and maintaining virus-specific CD8(+) effectors to contain HIV-1 in vivo. The results suggest that inactivated whole virus-pulsed DC vaccines could be a promising strategy for treating people with chronic HIV-1 infection. PMID 15568033

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells.
Juni 2004 | Carbonneil, Cédric; Saidi, Héla; Donkova-Petrini, Vladimira; Weiss, Laurence
Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy. PMID 15184347

Induction of anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) and CD4(+) T-cell reactivity by dendritic cells loaded with HIV-1 X4-infected apoptotic cells.
Feb. 2002 | Zhao, Xiao-Qing; Huang, Xiao-Li; Gupta, Phalguni; Borowski, Luann; Fan, Zheng; Watkins, Simon C; Thomas, Elaine K; Rinaldo, Charles R
T-cell responses to X4 strains of human immunodeficiency virus type 1 (HIV-1) are considered important in controlling progression of HIV-1 infection. We investigated the ability of dendritic cells (DC) and various forms of HIV-1 X4 antigen to induce anti-HIV-1 T-cell responses in autologous peripheral blood mononuclear cells from HIV-1-infected persons. Immature DC loaded with HIV-1 IIIB-infected, autologous, apoptotic CD8(-) cells and matured with CD40 ligand induced gamma interferon production in autologous CD8(+) and CD4(+) T cells. In contrast, mature DC loaded with HIV-1 IIIB-infected, necrotic cells or directly infected with cell-free HIV-1 IIIB were poorly immunogenic. Thus, HIV-1-infected cells undergoing apoptosis serve as a rich source of X4 antigen for CD8(+) and CD4(+) T cells by DC. This may be an important mechanism of HIV-1 immunogenicity and provides a strategy for immunotherapy of HIV-1-infected patients on combination antiretroviral therapy. PMID 11861866

Extracorporeal whole body hyperthermia treatment of HIV patients, a feasibility study.
Jan. 1998 | Zablow, A; Shecterle, L M; Dorian, R; Kelly, T; Fletcher, S; Foreman, M; Myers, R; Holton, M; Sanfilippo, L; St Cyr, J
The literature supports that the retrovirus, Human Immunodeficiency Virus (HIV), which is thought to cause Acquired Immunodeficiency Syndrome (AIDS), is heat sensitive at temperatures which can be achieved in man. Invasive or non-invasive induction of whole body hyperthermia (WBH) has been used to treat an array of illnesses, primarily in the field of oncology, until recently. Non-invasive methods have proven to be less toxic than invasive means. However, new technology and refined patient management have shown a dramatic decrease in the side effects with extracorporeal whole body hyperthermia (EWBH). The Food and Drug Administration granted a prospective trial for six HIV positive/AIDS patients to undergo a single treatment of EWBH, with patients randomized to a core temperature of either 41 or 42 degrees C. All patients had failed antiretrovirals and experienced at least one episode of an opportunistic infection. Organetics, Ltd., PS-1 extracorporeal, centrifugal pump device was used to induce EWBH. Results of this feasibility study demonstrated the ability of this equipment and technique to induce EWBH with acceptable toxicity. It was not possible to assess efficacy in this small study. PMID 9421740

Extracorporeal whole body hyperthermia treatments for HIV infection and AIDS.
Jan. 1998 | Ash, S R; Steinhart, C R; Curfman, M F; Gingrich, C H; Sapir, D A; Ash, E L; Fausset, J M; Yatvin, M B
Whole body hyperthermia therapy (WBHT) is the elevation of the core body temperature to 42 degrees C. In vitro studies have confirmed that 42 degrees C is cytocidal for virally infected lymphocytes, and even more effective when heating is repeated 4 days later. The safety and efficacy of two successive sessions of WBHT (4 days apart) was evaluated in 30 patients with AIDS (not on protease inhibitors), randomized to: 1) untreated controls, 2) low temperature WBHT for 1 hour at 40 degrees C and repeated 96 hours later, and 3) high temperature WBHT for 1 hour at 42 degrees C and repeated 96 hours later. The sorbent suspension in the ThermoChem System (HemoCleanse, West Lafayette, IN) system automatically controlled blood phosphate, calcium, and other electrolyte concentrations during WBHT. In 1 year of follow-up after WBHT, there were positive effects of the therapy on frequency of AIDS defining events, Karnofsky score, and weight maintenance. However, effects on plasma HIV RNA and CD4 counts were transient. Two successive WBHT treatments were performed in four patients who were on protease inhibitor/triple drug therapy, but had suboptimal response. In follow-up for 6 months, plasma HIV RNA and CD4 improved after WBHT, and the patients remained clinically well. This WBHT may have specific advantages in patients with suboptimal response to protease inhibitor therapy. PMID 9360163

Effect of whole-body hyperthermia on AIDS patients with Kaposi's sarcoma: a pilot study.
Mai 1996 | Steinhart, C R; Ash, S R; Gingrich, C; Sapir, D; Keeling, G N; Yatvin, M B
The safety and possible efficacy of extracorporeal whole-body hyperthermia (WBHT) were evaluated in the first FDA-approved feasibility study of WBHT in persons with AIDS. Six gay men, aged 20-50 years, CDC class C-3, underwent 1 h of WBHT at either 40 degrees C or 42 degrees C, employing a system that minimizes the physiological and biochemical changes that occur during WBHT. All subjects had Kaposi's sarcoma (KS), were free of opportunistic infections, and had significant elevations of plasma HIV RNA. During the treatment, there were no adverse side effects and all subjects tolerated WBHT without problems. KS lesions partially regressed immediately following WBHT in all subjects but returned to pretreatment status in five of six patients at 1 week. In subjects treated at 40 degrees C, CD4 counts decreased during the 8-week follow-up period; they remained unchanged, however, following 42 degrees C WBHT. Viral load remained unchanged following WBHT in subjects treated at 40 degrees C. Treatment at 42 degrees C resulted in an immediate reduction in HIV RNA that was not sustained at 1 week post-WBHT. We conclude that WBHT is safe in subjects with advanced HIV disease and that it may have a role in treating HIV infection. A larger controlled trial involving two treatments in less immunocompromised subjects is currently in progress to test this hypothesis. PMID 8603263

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