Hélène Le Buanec

Vaccination with Tat toxoid attenuates disease in simian/HIV-challenged macaques

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo. We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-alpha, and chemokine receptor expression (CXCR4 and CCR5) on CD4(+) T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation.

The proinflammatory cytokine TNFα is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohns disease. As an alternative to anti-human TNFα (hTNFα) mAbs and other hTNFα blocker approved drugs, we developed an active anti-hTNFα immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFα heterocomplex immunogen (hTNFα kinoid) adjuvanted in incomplete Freunds adjuvant. In mice transgenic for hTNFα (TTg mice), hTNFα kinoid vaccination elicited high titers of Abs that neutralized hTNFα bioactivities but did not result in a cellular response to hTNFα. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFα-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFα exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

IFNα kinoid vaccine-induced neutralizing antibodies prevent clinical manifestations in a lupus flare murine model

A major involvement of IFNα in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNα from a recombinant adenovirus (IFNα Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) × New Zealand White (NZW)F1 mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNα immunogen, termed IFNα kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNα Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNα inhibitory capacity was present in the serum.

VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases

Tumor growth depends on blood supply, requiring the development of new vessels, and vascular endothelial growth factor (VEGF) plays a central role in neoangiogenic processes. For this reason, VEGF represents a target for the development of new therapeutic antiangiogenic molecules. Clinical trials using anti-VEGF mAbs such as bevacizumab have validated the efficacy of this therapeutic approach but have also revealed adverse effects. Here we report that a VEGF-derived immunogen, consisting of a heterocomplex of a murine (m)VEGF and keyhole limpet hemocyanin, called “mVEGF kinoid,” triggered a strong Ab immune response in mice. The anti-VEGF Abs inhibited both the proliferation of human umbilical vein endothelial cells cultured in the presence of mVEGF and the binding of mVEGF to its receptor-2 Flk-1. In mVEGF kinoid-immunized BALB/c mice challenged with syngeneic CT26 colorectal tumor cells, the number and size of lung metastases were significantly decreased. In human (h)VEGF kinoid-immunized BALB/c mice, high levels of serum Abs to hVEGF were present, and purified IgG from these mice decreased by ≥50% the tumor growth of human A673 rhabdomyosarcoma cells and HT29 colon carcinoma xenografted in Swiss nude and NOD/SCID mice, respectively. Tumor cell growth inhibition was similar to that observed in mice receiving therapeutic doses of bevacizumab. These experiments suggest that a therapeutic vaccine containing VEGF kinoid may represent a strategy for safely combating VEGF-dependent neovascularization and metastases occurring in malignant tumors.

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection

Significance Our candidate HIV vaccine, a single-chain gp120-CD4 chimera, elicits protection against acquisition of simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) in rhesus macaques. Antibody-dependent cellular cytotoxicity was an inverse correlate of infection risk. However, it is attenuated when antigen-specific T-cell responses exceed a threshold, presumably due to the generation of CD4+ CCR5+ T cells, the preferred cellular targets of SHIV/SIV. Multiple studies strongly suggest that HIV/SIV-specific T-cell responses are a double-edged sword. On one hand, they are required for T-cell help in the protective antibody response. On the other hand, they appear to mitigate protection by creating new targets for viral replication. Determining the balance between protective antibody responses and attenuating T-cell responses is a key challenge confronting HIV vaccine development. A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

CD24 hi CD27 + and plasmablast-like regulatory B cells in human chronic graft-versus-host disease

Interleukin 10 (IL-10)-producing B cells (regulatory B cells [Bregs]) regulate autoimmunity in mice and humans, and a regulatory role of IL-10-producing plasma cells has been described in mice. Dysfunction of B cells that maintain homeostasis may play a role in the pathogenesis of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation. Here, we found a relation between decreased Breg frequencies and cGVHD severity. An impaired ability of B cells to produce IL-10, possibly linked to poor signal transducer and activator of transcription 3 and extracellular signal-regulated kinase phosphorylation, was found in patients with active cGVHD. IL-10 production was not confined to a single B-cell subset, but enriched in both the CD24(hi)CD27(+) and CD27(hi)CD38(hi) plasmablast B-cell compartments. In vitro plasmablast differentiation increased the frequency of IL-10-producing B cells. We confirmed that allogeneic transplant recipients had an impaired reconstitution of the memory B-cell pool. cGVHD patients had less CD24(hi)CD27(+) B cells and IL-10-producing CD24(hi)CD27(+) B cells. Patients with cGVHD had increased plasmablast frequencies but decreased IL-10-producing plasmablasts. These results suggest a role of CD24(hi)CD27(+) B-cell and plasmablast-derived IL-10 in the regulation of human cGVHD.

Tat toxoid as a component of a preventive vaccine in seronegative subjects

Because administration of Tat protein, the HIV-1 toxin that induces immunosuppression and apoptosis, may be deleterious to the host immune system, a chemically inactivated but nonetheless immunogenic Tat preparation, Tat toxoid, was used to immunize seronegative individuals against Tat. In an open, controlled, phase I clinical trial, Tat toxoid turned out to be safe, well tolerated, and able to trigger a specific immune reaction. In particular, a threefold to more than 10-fold increase of circulating antibodies directed against the native Tat was observed after immunization in all of 5 immunized study subjects, together with a positive reaction to delayed-type hypersensitivity (DTH) skin test with Tat toxoid in vivo and increased lymphoproliferative response to native Tat in vitro. Persistent (> or =1 year) high levels of circulating anti-Tat antibodies could prevent the Tat-induced immune suppression and, following HIV-1 exposure, allow the anti-HIV-1 cellular immune response, with its early release of protective beta-chemokines, to occur leading to an increase of host resistance, that is, protection.

IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients.

Identification of immunodominant epitopes in inactivated Tat-vaccinated healthy and HIV-1-infected volunteers.

We analyzed the epitopes and the molecular forms of Tat recognized by the antibodies raised by Tat-toxoid vaccination in both healthy and HIV-infected volunteers. Tat-toxoid-vaccinated healthy volunteer sera reacted predominantly with peptides covering amino acids 1 through 24 and 46 through 60, corresponding to the N-terminus and basic domains of Tat. In contrast, whereas all sera from vaccinated HIV-1-positive patients reacted with the N-terminus and (with a single exception) with the basic domain, most of these sera also recognized peptides encompassing distinct domains of Tat, particularly the C-terminus (79-86). The sera of vaccinated individuals recognized both monomeric and oligomeric forms of Tat 1 through 86 or of Tat 1 through 101 and also blocked the ability of cell-released extracellular Tat to transactivate the HIV-1 LTR promoter. Synthetic Tat preincubated with sera from vaccinated individuals lost its functional activity as well. This is probably because of its inability to enter the cells as a result of immune complex formation with anti-Tat IgG. These data demonstrate that Tat-toxoid vaccination induces an efficient antibody response blocking the functional activity of Tat.

Purification and Immunophenotypic Characterization of Human B Cells with Regulatory Functions

The analysis of human B cell populations of the blood relies on the expression of surface markers, mainly CD19, CD24, CD38, and CD27. According to these surface markers, three main B cell subsets can be identified in the blood: immature transitional B cells (CD19(+)CD24(high)CD38(high)), naïve B cells (CD19(+)CD24(int)CD38(int)) that have not encountered an antigen, and memory B cells (CD19(+)CD27(+)). To date, human B cells with regulatory functions have been essentially described within the CD24(high)CD38(high) transitional B cell subset. CD24(high)CD38(high) transitional B cells are able to produce interleukin 10 (IL-10) and to regulate in vitro Th1 and Th17 CD4(+) T cell activation. Here, we provide the methods to analyze and purify the CD24(high)CD38(high) transitional B cell subset for further in vitro experiments. We also provide a reliable method to detect B cell IL-10 production using intracellular cytokine staining.