A reduction of viral mRNA, proteins and induction of altered morphogenesis reveals the anti-HTLV-1 activity of the labdane-diterpene myriadenolide in vitro.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) has been associated with leukemia/lymphoma (ATL) and myelopathy/tropical spastic paraparesis (HAM/TSP), in addition to other inflammatory diseases as well as infection complications. Therapeutic approaches for HTLV-1-related pathologies are limited. The labdane diterpene myriadenolide (AMY) is a natural product that exhibit biological activities, such as anti-inflammatory and antiviral activity as reported for HIV and herpesvirus. RESULTS: We demonstrated that this natural product was able to inhibit the expression of gag-pol mRNA and substantially reduced the expression of the structural proteins p19 and gp46. Comparison of treated and untreated cells shows that AMY alters both the morphology and the release of viral particles. The Atomic Force Microscopy assay showed that the AMY treatment reduced the number of particles on the cell surface by 47%. CONCLUSION: We demonstrated that the labdane diterpene myriadenolide reduced the expression of the structural proteins and the budding of viral particles, besides induces altered morphogenesis of HTLV-1, conferring on AMY a new antiviral activity that may be useful for the development of new compounds with specific anti-HTLV-1 activity.

Anti-Tax antibody levels in asymptomatic carriers, oligosymptomatic carriers, patients with rheumatologic disease or with HAM/TSP do not correlate with HTLV-1 proviral load.

BACKGROUND: HTLV-1 infects millions of people around the world and induces myelopathy (HAM/TSP), adult T-cell leukemia (ATL) or other inflammatory or rheumatologic diseases. The host-virus interaction causes asymptomatic carriers to develop HAM/TSP. Biomarkers are needed to predict patients who are at risk for HAM/TSP. Tax is highly immunogenic and is a major target protein recognized by cytotoxic T lymphocytes. Anti-Tax antibodies are involved in HAM/TSP pathogenesis. OBJECTIVES: To assess anti-Tax IgG reactivity with a flow cytometry assay (FCA) using an infection/transfection system with Vaccinia virus and pLW44/Tax-expressing Tax and to correlate the anti-Tax response and the HTLV-1 proviral load. STUDY DESIGN: : We enrolled 81 individuals: 9 HTLV-1 seronegative (NP) and 72 HTLV-1 positive (23 HTLV-1 asymptomatic carriers (AC), 12 oligosymptomatic patients (OL), 7 with rheumatologic diseases (DR) and 30 with HAM/TSP (HT)). Anti-Tax reactivity was assessed by FCA, and HTLV-1 proviral load was measured with real time PCR. RESULTS: The HT and DR groups showed greater anti-Tax IgG reactivity (p<0.001 and p<0.05 comparing HT to the OL and AC group, respectively; p<0.05 comparing DR to the OL group), and the reactivity in the DR+HT group was significantly different when compared to the AC group (p<0.05) and to the OL group (p<0.001). The proviral load was higher in the HT group compared to the OL (p<0.001) and in the HT+DR group compared to OL (p<0.001). There was no correlation between anti-Tax IgG reactivity and proviral load in any of the HTLV-1-infected groups. CONCLUSION: These findings suggest that although anti-Tax IgG reactivity and the HTLV-1 proviral load are important markers of the development of HTLV-1-associated diseases, their levels are not correlated.

Diagnostic tool based on an HTLV-1-Tax expression system in eukaryotic cells using a poxvirus vector.

Human T-lymphotropic virus 1 (HTLV-1) induces an immune-mediated inflammatory disease affecting the nervous system that eventually is accompanied by ocular, rheumatic and dermatologic manifestations (HTLV-1 associated myelopathy/tropical spastic paraparesis, or HAM/TSP). Proviral load and HTLV-1 protein expression, mainly of Tax, is correlated with disease progression and induction of host-virus equilibrium breakdown that, reportedly, involves the presence of Tax-specific cytotoxic T lymphocytes (CTL), T regulatory cells and anti-Tax antibodies. Based on knowledge of anti-Tax antibodies as markers of disease progression, the objectives of this study were both to design an infection/transfection system using the Vaccinia virus and a tax-encoding plasmid for the expression of Tax protein as well as to use this cell support to evaluate anti-Tax IgG by flow cytometry. The flow cytometry assay was standardized using pooled sera from each test group (negative, asymptomatic and HAM/TSP patients). The HAM/TSP group presented higher IgG anti-Tax reactivity (above 70%) than the asymptomatic group (nearly 40% reactivity). The data indicate that the infection/transfection system is useful for assessing Tax expression. This is a promising assay for use as a diagnostic tool to detect IgG anti-Tax and monitor HTLV-1 infected individuals.

Performance of IgG and IgG1 anti-HTLV-1 reactivity by an indirect immunofluorescence flow cytometric assay for the identification of persons infected with HTLV-1, asymptomatic carriers and patients with myelopathy.

In this study, the performance of IgG and IgG1 anti-HTLV-1 reactivity obtained by a flow cytometric assay was evaluated to verify its applicability for the diagnosis of persons infected with HTLV-1, including asymptomatic carriers and patients with myelopathy. The ability to identify patients with myelopathy among persons infected with HTLV-1 was also examined. Western blot assays were performed to assess the reactivity profiles of sera from asymptomatic carriers and patients with myelopathy against viral proteins. The data showed that IgG1 detected by flow cytometric assay is effective for the diagnosis of persons infected with HTLV-1 with 97% sensitivity and 100% specificity. IgG and IgG1 exhibited high performance in distinguishing patients with myelopathy from asymptomatic carriers. Using serum dilutions and cut-off points established previously a second HTLV-1 carrier group was tested using flow cytometric assay to detect IgG and IgG1. The data demonstrated sensitivity of 93% and 98%, respectively, confirming the high reactivity of persons infected with HTLV-1 detected by this method. Western blot assays confirmed the high specificity of MT-2 cells as a reliable source of viral antigen since only sera from persons infected with HTLV-1 recognised MT-2 proteins. Furthermore, a high reactivity to Gag and Env proteins was observed, especially among patients with myelopathy. These data suggest that flow cytometric detection of IgG1 is a valuable, non-conventional serological method to diagnose HTLV-1 infection and for research purposes.