Title: In vitro and ex vivo evaluation of currently used and novel therapeutic strategies in HTLV-1 infection
Other Titles: In vitro en ex vivo evaluatie van huidige en nieuwe anti-HTLV-1 therapeutische strategieën
Authors: Moens, Britta; M0216962;
Issue Date: 7-Jun-2012
Abstract: <objectIsolated in 1979, the human T-lymphotropic virus type 1 (HTLV-1) was the first human pathogenic retrovirus to be discovered. In the meantime, HTLV-1 is found worldwide, with an estimated 10 to 30 million infected individuals, and endemic regions include Sub Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia/Australia. The majority of HTLV-1-infected individuals remain clinically asymptomatic throughout their lifetimes, while 5-10 % develops clinical manifestations, of which the two best-characterized diseases are HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). HAM/TSP is a neurological disease, for which treatment consists of mainly symptomatic and empirical strategies such as the use of immunosuppressive drugs, interferon-alpha (IFN-a), vitamin C (ascorbic acid, AA) or supportive care with antispasmodics. However, due to the shortcomings in clinical therapeutic trials, further studies are warranted to support the value of one of these specific treatment approaches over others. HTLV- 1-infected carriers can also develop ATLL, an aggressive T cell malignancy of mainly CD4+ CD25+ T cells. First line therapy for leukemic ATLL patients consists of the combination of AZT + IFN-a, whereas chemotherapy is administered to lymphoma ATLL patients. Nevertheless, intrinsic chemoresistance of malignant ATLL cells, relapse of patients, hypercalcaemia, opportunistic infections together with the lack of alternative efficient therapeutic strategies, all contribute to a very poor prognosis. Despite the severity of these diseases, there is no vaccine, no efficient therapy nor standardized marker to determine the risk of pathogenesis or prognosis in HTLV-1-infected individuals. Due to the failure to identify an unambiguous prognostic marker for HTLV-1 infection and/or associated diseases, the proviral load (PVL) applies as the most accepted surrogate marker for HTLV-associated disease progression, despite a lack of solid evidence of its value and validated techniques to measure PVL. Considering the number of asymptomatic HTLV-1-infected individuals and the poor prognosis of HAM/TSP and ATLL patients, the main aim of this doctoral thesis is to contribute to a better quantification of PVL and a better understanding of currently used and novel therapeutic anti HTLV 1 strategies.Since the discovery of HTLV-1, three other genotypes and ten subtypes have been recognized. Quantitative PCR (qPCR) techniques have been developed for detection of HTLV and quantification of PVL. However, the majority of published HTLV qPCR assays are singleplex assays and thus not capable of detecting the growing diversity of HTLV genotypes and subtypes. In addition, none of the reported qPCR assays have been compared with each other through quality control programs. To this purpose, part of this doctoral thesis was aimed at developing and evaluating a multiplex qPCR for simultaneous detection, genotyping and quantification of PVL of HTLV-1, 2 and 3. We demonstrate the broad dynamic range, specificity, and efficiency on genotyping, subtyping and PVL determination of our triplex assay in 163 infected specimens. HTLV-1 PVL levels were significantly higher in both ATLL and HAM/TSP patients in comparison with asymptomatic carriers (ACs), a finding consistent with the literature. In addition, comparison of our triplex qPCR with a previously published HTLV-1 singleplex qPCR, developed at a different institute (Lima, Peru), revealed excellent correlation between HTLV-1 PVL values determined with both assays, indicating the reliability and relevance of both techniques. We concluded that our novel assay offers an accurate test for molecular diagnosis and genotyping together with the determination of the PVL of HTLV-infected individuals, all in a single amplification reaction. In addition, we underlined the importance and the necessity of the validation of currently implemented HTLV qPCR assays in order to standardize these techniques. In this way, reliable and reproducible quantification of the HTLV PVL surrogate marker can be assured, which will improve both the evaluation of HTLV associated disease progression and treatment success.In a second part of this doctoral thesis, we questioned the relevance of IFN-a in the therapeutic management of HTLV-1-infected individuals through an ex vivo and in vitro evaluation of the effects of IFN-a. Based on observational studies in HAM/TSP patients, clinical benefit has been ascribed to IFN-a on the one hand, the use of which is characterized by substantial side effects, and to high-dose AA on the other hand, a drug with mild side effects. We report for the first time superior antiproliferative, cell death-inducing and immunomodulatory effects of high-dose AA over IFN-a in HAM/TSP peripheral blood mononuclear cells (PBMCs) ex vivo, and HTLV-1-infected T-cell lines in vitro. In addition, we identified that AA treatment modulated cell death-associated pathways, suggesting that the significant antiproliferative and immunomodulatory effects of high-dose AA might be due to elimination of HTLV-1-infected cells. Due to the limited number of HAM/TSP clinical trials, additional studies are still necessary to determine the clinical value of high-dose AA in HAM/TSP treatment. Nevertheless, our findings already reveal ex vivo molecular mechanisms of action for high-dose AA and thus provide a rationale for its clinical use in HAM/TSP treatment. In addition, controversy exists with regard to the mechanism of action of IFN-a in HTLV-1 infection, fuelled by in vitro evidence of HTLV-1-induced blunting of IFN-a signaling pathways. We therefore addressed the possible antiviral, antiproliferative and/or pro-apoptotic effects of IFN-a in HTLV-1 mono-infected and human immunodeficiency virus type 1 (HIV-1)/HTLV-1 co-infected CD4+ T-cell lines. IFN-a exerted pronounced anti-HIV-1 effects in HTLV-1 co-infected cells, but had a limited, post-transcriptional antiviral effect directed against HTLV-1 in mono-infected cells. As demonstrated by gene expression profiling, both STAT1/STAT2 antiviralsignaling pathways and broad interferon stimulated genes (ISGs) were fully activated by IFN-a in HTLV-1-infected cells. However, IFN-a failed to inhibit mRNA levels of any of the HTLV-1 genes or to induce significant pro-apoptotic or antiproliferative activity in HTLV-1-infected cells. We concluded that the apparent absence of IFN-a antiretroviral activity was selective towards HTLV-1, since a significant anti-HIV-1 effect was observed in co-infected cells.Despite the controversy regarding the exact mechanism of action of IFN-a in HTLV-1 infection, first-line therapy for ATLL includes IFN-a treatment, given that AZT + IFN-a combination therapy represents the principal anti-ATLL treatment. However, few data exist regarding the mechanism of action of AZT + IFN-a combination therapy. Therefore, we evaluated the effects of combination treatment in HTLV-1-infected cell lines. We confirmed the previously described absence of cytotoxicity of combination treatment in MT-2 cells. In contrast, AZT + IFN-a exerted a synergistic effect with both pro-apoptotic and antiproliferative activity in MT-4 cells, without evidence for distinct antiviral activity. In addition, multiple cellular pathways associated with cell survival and cell death were involved in the in vitro mechanism of action of AZT + IFN-a. We speculated that the extent of activation of cell death pathways, and not the IFN-a-induced pathways, determines the synergistic effect of AZT + IFN-a in vitro and in vivo, as such association with clinical response has already been reported in vivo. Despite the above-mentioned therapeutic strategies, prognosis of HAM/TSP and ATLL patients remains poor. Nevertheless, only few novel candidate compounds are evaluated for HAM/TSP or ATLL, of which the majority is associated with significant toxicity. Given the necessity to improve therapy, we performed an in vitro and ex vivo comparison of candidate compounds and compounds currently implemented in HAM/TSP or ATLL treatment. We identified a promising pro-apoptotic, antiviral and immunomodulatory candidate compound, Q3, in HTLV-1-infected cell lines. Furthermore, the Q3 candidate compound exerted superior biological activity in comparison to the “gold standard” AZT + IFN-a combination treatment. We concluded that Q3 monotherapy can become an attractive therapeutic strategy, particularly given that Q3 is a derivative of a class of compounds already in clinical use for other medical applications for more than two decades, suggesting tolerable in vivo side effects.We conclude that both the evaluation of PVL assays as well as currently used and novel anti-HTLV-1 compounds, will contribute to improved clinical management of HTLV-associated pathologies.</object
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Rega Institute for Medical Research (-)
Laboratory of Virology and Chemotherapy (Rega Institute)
Laboratory of Clinical and Epidemiological Virology (Rega Institute)

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