Browsing by Author "Tomasicchio, Michele"
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- ItemOpen AccessDevelopment of an Autologous Human Dendritic Cell Vaccine against Mycobacterium tuberculosis in Patients with Extensively Drug-Resistant Tuberculosis(2022) Londt, Rolanda Sabrina; Dheda, Keertan; Tomasicchio, MicheleIntroduction: Extensively drug-resistant tuberculosis (XDR-TB), and resistance beyond XDR-TB (often untreatable), is an increasing public health concern globally. Drug resistance has outpaced the drug development pipeline. Therefore, alternative immunotherapeutic approaches are urgently needed. In this proof-of-concept study, and based on precedents in breast and prostate cancer, we aimed to develop an autologous therapeutic dendritic cell (DC) vaccine by evaluating two TB antigen-specific multi-peptide pools in combination with different adjuvants (such a cellular vaccine would require ex-vivo manipulation in a clean room and reinfusion back into the patient). Vaccine efficacy was evaluated using an in vitro mycobacterial containment model. Methods: DCs were derived from monocytes isolated from the peripheral blood of patients with XDR-TB (n=30) and participants with presumed latent TB infection (LTBI; n=15). DCs were matured with a differential combination of cytokines and pattern-recognition receptor agonists (maturation cocktail) together with different TB antigen combinations. The complete cocktail contained interferon-, interferon-, CD40L, IL-1 and TLR-3, TLR-7 and TLR-8 agonists, whilst in the limited cocktail the TLR agonists were absent. Two M.tb-specific multi peptide pools suited to GMP-grade vaccine manufacture were evaluated: (i) an immunodominant peptide pool (ESAT6 + CFP10 + Ag85B + TB10.4; referred to as ECAT) and (ii) a PE/PPE peptide pool. PPD and the lysate of a clinical M.tb strain (HN878), though not amenable to GMP-grade vaccine development, served as controls representing the entire antigenic repertoire of M.tb. Thus, there were four major comparator groups (unstimulated DCs, limited cocktail-only stimulated DCs, antigen-only stimulated DCs, and antigen + complete cocktail stimulated DCs). As the two latter groups were interrogated using the two multi-peptide pools and the two broad-spectrum antigen controls, a total of 10 different experimental groups were generated (see overview figure 3.2). DCs were assessed for the expression of key maturation markers using flow cytometry and the secretion of Th1-polarising cytokines by ELISA. The ability of DC-primed peripheral blood mononuclear cells (PBMCs) to restrict the growth of M.tb-infected monocyte derived-macrophages was evaluated using an in vitro validated mycobacterial containment assay. Results: In patients with XDR-TB, DCs matured with any M.tb-antigen + complete cocktail, compared to DCs matured with M.tb-antigen only, showed significantly higher upregulation of CD80, CD83, CD86, and CCR7 (p< 0.001 for all comparisons), and higher secreted levels of IL12p70 (0.67 versus 0.01 ng/mL per 106 cells; p< 0.001). A similar pattern was seen in the containment experiments: mycobacterial stasis within the XDR-TB group was significantly better with antigen + complete cocktail versus antigen alone (p≤0.0002 for PE/PPE and PPD), and the limited cocktail did not show this effect. Furthermore, PE/PPE + complete cocktail matured DCs achieved a higher magnitude of mycobacterial containment compared to ECAT + complete cocktail-matured DCs (50%, IQR:39-75, versus 46%, IQR: 15-62, p= 0.02). Using PPD and the HN878 lysate did not improve the containment effect. Furthermore, the improved containment effect of the PE/PPE + complete cocktail, versus ECAT + complete cocktail, was only seen in the XDR-TB and not in the LTBI group. Conclusion: In patients with XDR-TB, an effector response primed by PE/PPE antigen and complete cocktail-matured DCs was able to better restrict the growth of M.tb in vitro. These data indicate proof-of-concept feasibility to generate a DC-based immunotherapeutic intervention for therapeutically destitute patients with DR-TB. Further mechanistic studies and future phase 1 and 2 human clinical studies are warranted.
- ItemOpen AccessThe progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor(Public Library of Science, 2013) Tomasicchio, Michele; Avenant, Chanel; Toit, Andrea Du; Ray, Roslyn M; Hapgood, Janet PThe glucocorticoid receptor (GR) regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr), can modulate the transcriptional response of the GR. Glucocorticoids (GCs) and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA) is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A). We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4 + T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex), cortisol and MPA induce apoptosis in primary CD4 + T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4 + T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4 + T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to susceptibility and progression of diseases such as HIV-1.