Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

Since human T-lymphotropic virus type 1 (HTLV-1)-associated diseases are associated with a high HTLV-1 load, reducing this load may treat or prevent disease. However, despite in vitro evidence that certain nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are active against HTLV-1, in vivo results have been disappointing. We therefore assayed the sensitivity of HTLV-1 primary isolates to a panel of RT inhibitors. HTLV-1 primary isolates were obtained, pre- and post- NRTI treatment, from patients with HTLV-1-associated myelopathy. Sensitivity to azidothymidine (AZT), lamivudine (3TC), tenofovir (TDF) and three phosphonated carbocyclic 2’-oxa-3’aza nucleosides (PCOANs) was assessed in a RT inhibitor assay. With the exception of 3TC, HTLV RT from primary isolates was less sensitive to all tested inhibitors than HTLV-1 RT from MT-2 cells. HTLV-1 RT from primary isolates and from chronically infected, transformed MT-2 cells was insensitive to 3TC. Sensitivity of primary isolates to RT inhibitors was not reduced following up to 12 months of patient treatment with AZT plus 3TC. The sensitivity of HTLV-1 primary isolates to NRTIs differs from that of cell lines and may vary among patients. Failure of NRTIs to reduce HTLV-1 viral load in vivo was not due to the development of phenotypic NRTI resistance. AZT and the three PCOANs assayed all consistently inhibited primary isolate HTLV-1 RT

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

Abstract: Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset

HLA Class I Binding of HBZ Determines Outcome in HTLV-1 Infection

CD8(+) T cells can exert both protective and harmful effects on the virus-infected host. However, there is no systematic method to identify the attributes of a protective CD8(+) T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8(+) T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8(+) T cell response through to the least effective CD8(+) T cell response. We conclude that CD8(+) T cells play an important role in the control of HTLV-1 and that CD8(+) cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8(+) T cell response

The cellular immune response to the HTLV-1 protein HBZ

associated myelopathy (HAM). The pathogenesis of HAM is not fully understood. However, considerable evidence points to the importance of the regulatory protein Tax. Tax promotes the proliferation of HTLV-1 infected cells but also induces a strong cytotoxic-T-lymphocyte response. The contribution of the HTLV-1 basic leucine zipper protein (HBZ), which promotes T cell proliferation but suppresses Tax mediated transactivation, remains to be elucidated. This thesis explores the following hypotheses:- 1. HBZ is expressed in vivo and will elicit an immune response in HTLV-1 infected subjects. 2. HBZ mRNA will be detected in peripheral blood mononuclear cells (PBMCs) from HTLV-1 infected subjects. To test these hypotheses 48 subjects: 14 patients with HAM, 28 asymptomatic carriers (ACs) (14 with high viral load >1% and 14 with low viral load <1%), and six HTLV-1 negative individuals were studied. PBMCs and plasma were isolated from whole blood and preserved for future use. The first hypothesis was addressed by developing HBZ and Tax enzyme linked immuno spot assays (ELIspot) with overlapping peptides that span the entire proteins. The frequency of Tax-specific and HBZ-specific CD4+ and CD8+ T cells secreting IFN- or IL-2 were compared. The presence of any HBZ-specific CD8+ IL-2 secreting T cell response was most commonly detected in ACs with low viral load. Tax-specific CD8+ IL-2 secreting T cell responses were most commonly detected in patients with HAM but where detected the number of IFN- secreting Tax-specific CD8 cells was highest in patients with HAM. No significant difference in CD4+ T cell responses was observed. The second hypothesis was tested by developing real-time PCR to detect and quantify HBZ and Tax mRNA. HBZ mRNA was detected in PBMCs from patients with high viral load regardless of disease whilst Tax mRNA was only detected in three patients, all with HAM. HTLV-1 viral load correlated positively with HBZ mRNA, but not Tax. At this point, two new hypotheses were considered: - 3. The cytokine profile differs between patients with HAM and ACs. 4. Cytokine profile and ELIspot frequency will be altered during in vivo treatment with immunomodulators.The cytokine profile was examined in a subset of 12 patients using a commercial electrochemiluminescence assay that simultaneously quantifies nine proinflammatory cytokines. GM-CSF, IL-12-p70 and IL-8 were disease associated. IFN-, IL10, IL-6 and TNF- were viral load dependent. IL-1and IL-2 did not discriminate. In vivo treatment with Infliximab and Ciclosporin A did not significantly alter the cytokine profiles. In conclusion, a detectable HBZ response is associated with an absence of HBZ mRNA and is most common in AC with low viral load. The absence of HBZ responses is associated with detectable HBZ mRNA and the presence of Tax responses and is associated with high viral load and the presence of HAM.Open acces