HTLV-1 Proviral Load Research Articles

Long-term follow-up of HTLV-1 proviral load in asymptomatic carriers and in incident cases of HAM/TSP: what is its relevance as a prognostic marker for neurologic disease?

HTLV-1 proviral load (pvl) is an important risk marker for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but its value as prognostic marker is not well defined. Long-term prospective cohort studies are necessary to clarify this question. Here, we analyzed HTLV-1 pvl in the peripheral blood of 82 asymptomatic carriers (AC; 351 samples), 12 HAM/TSP patients (HAM; 46 samples), and six incident cases of HAM/TSP (iHAM), with serial samples collected before (n=10) and after (n=20) the disease onset. The mean interval of follow-up was 10years in the AC group and 8years in HAM and iHAM groups. pvl was not significantly different between the first and last measurements in the three groups, but there was a trend to decrease over time. Coefficient of variation of pvl was significantly lower in the AC group than in HAM (p=0.015) and iHAM (p=0.022) patients. AC and HAM individuals showed a significant and strong positive correlation between the first and last measurements of pvl, but not iHAM subjects. All individuals who developed HAM/TSP during the follow-up had high pvl level (>1%) before the onset of disease, but a typical increase in pvl was not observed in that period. The data suggest that there is a trend to reach an equilibrium plateau of pvl over time, characteristic of each individual. A significant rate of AC keeps high pvl levels for a long time without developing clinical symptoms associated to HTLV-1 infection. Thus, serial quantification of pvl in the peripheral blood does not seem to be a good prognostic marker for HAM/TSP.

Evidence of Brain Inflammation in Patients with Human T-Lymphotropic Virus Type 1-Associated Myelopathy (HAM): A Pilot, Multimodal Imaging Study Using 11C-PBR28 PET, MR T1-Weighted, and Diffusion-Weighted Imaging.

Five subjects with HAM and 2 HTLV-1 asymptomatic carriers were studied. All underwent clinical neurologic assessment including cognitive function and objective measures of gait, quantification of HTLV-1 proviral load in peripheral blood mononuclear cells, and human leukocyte antigen-antigen D related expression on circulating CD8+ lymphocytes. 11C-PBR28 PET and MRI were performed on the same day. 11C-PBR28 PET total volume of distribution and distribution volume ratio (DVR) were estimated using 2-tissue-compartment modeling. MRI data were processed using tools from the FMRIB Software Library to estimate mean diffusivity (MD) and gray matter (GM) fraction changes. The results were compared with data from age-matched healthy volunteers. Across the whole brain, the total volume of distribution for the subjects with HAM (5.44 ± 0.84) was significantly greater than that of asymptomatic carriers (3.44 ± 0.80). The DVR of the thalamus in patients with severe and moderate HAM was higher than that in the healthy volunteers, suggesting increased translocator protein binding (z > 4.72). Subjects with more severe myelopathy and with high DR expression on CD8+ lymphocytes had increased DVR and MD (near-significant correlation found for the right thalamus MD: P = 0.06). On the T1-weighted MRI scans, the GM fraction of the brain stem was reduced in all HTLV-1-infected patients compared with controls (P < 0.001), whereas the thalamus GM fraction was decreased in patients with HAM and correlated with the disease severity. There was no correlation between neurocognitive function and these markers of central nervous system inflammation. This pilot study suggests that some patients with HAM have asymptomatic inflammation in the brain, which can be detected and monitored by 11C-PBR28 PET together with structural and diffusion-weighted MRI.

HLA-G 3'-untranslated region polymorphisms are associated with HTLV-1 infection, proviral load and HTLV-associated myelopathy/tropical spastic paraparesis development.

Most human T-lymphotropic virus type 1 (HTLV-1)-infected patients remain asymptomatic throughout life. The factors associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development have not been fully elucidated; immunological and genetic factors may be involved. The association of 14 bp INS/DEL HLA-G polymorphism with HTLV-1 infection susceptibility has been reported previously. Here, other polymorphic sites at the HLA-G 3'-UTR (14-bp D/I, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G and +3196C/G) were evaluated in 37 HTLV-1-infected individuals exhibiting HAM/TSP, 45 HTLV-1 asymptomatic carriers (HAC) and 153 uninfected individuals, followed up at University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. It was observed that: (i) 14bpDI genotype is a risk factor for HTLV-1 infection, while the 14bpDD and +3142CC genotypes were associated with protection against infection; (ii) the +3142C allele and the +3003CT and +3142CC genotypes were associated with susceptibility, while 14bpII and +3003TT genotypes were associated with protection against HAM/TSP development; and (iii) the 14bpII, +3010CC, +3142GG and +3187AA genotypes were associated with lower HTLV-1 proviral load compared to respective counterpart genotypes. Findings that HLA-G has a well-recognized immunomodulatory role and that the genetic variability at HLA-G 3'-UTR may post-transcriptionally modify HLA-G production indicate a differential genetic susceptibility to: (i) the development of HTLV-1 infection, (ii) the magnitude of HTLV-1 proviral load and (iii) HAM/TSP development.

Effect of Pulsed Methylprednisolone on Pain, in Patients with HTLV-1-Associated Myelopathy.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an immune mediated myelopathy caused by the human T-lymphotropic virus type 1 (HTLV-1). The efficacy of treatments used for patients with HAM/TSP is uncertain. The aim of this study is to document the efficacy of pulsed methylprednisolone in patients with HAM/TSP. Data from an open cohort of 26 patients with HAM/TSP was retrospectively analysed. 1g IV methylprednisolone was infused on three consecutive days. The outcomes were pain, gait, urinary frequency and nocturia, a range of inflammatory markers and HTLV-1 proviral load. Treatment was well tolerated in all but one patient. Significant improvements in pain were: observed immediately, unrelated to duration of disease and maintained for three months. Improvement in gait was only seen on Day 3 of treatment. Baseline cytokine concentrations did not correlate to baseline pain or gait impairment but a decrease in tumour necrosis factor-alpha (TNF-α) concentration after pulsed methylprednisolone was associated with improvements in both. Until compared with placebo, treatment with pulsed methylprednisolone should be offered to patients with HAM/TSP for the treatment of pain present despite regular analgesia.

Higher HTLV-1c proviral loads are associated with blood stream infections in an Indigenous Australian population

BackgroundAn association between blood stream infections (BSI) and HTLV-1 seropositivity in Indigenous Australians might result from HTLV-1 mediated inflammation and parasite coinfections that provide portals of entry for bacteria. ObjectivesTo determine whether BSI risk increases with HTLV-1c proviral load (PVL) and to identify the pathogens responsible in the context of HTLV-1 related conditions. Study designIndigenous adults admitted to Alice Springs Hospital, central Australia, were recruited as cases or controls according to whether they had a BSI. Clinical data were extracted from case notes and the hospital pathology database. HTLV-1 serology and PVL assays were then performed and risk factors for BSI were determined for HTLV-1 infected subjects. ResultsRisk factors were compared between 44 cases and 30 controls who were HTLV-1 Western blot positive. In a multivariable model, high HTLV-1c PVL was predictive of BSI during the study period (OR, 9.10; 95% CI, 2.40–34.4). Median (IQR) HTLV-1c PVL (copies per 100 PBL) was 39-fold higher for patients recording any BSI (0.116 (0.009, 0.277)) relative to those who had no BSI (0.003 (0.000, 0.067))(p=0.0007). Mean (SD) PVL for subjects with no BSI (n=27), 1 BSI (n=37) and ≥2 BSI (n=10) during the study period were 0.120 (0.301), 0.271 (0.622) and 0.500 (0.654) copies per 100 PBL, respectively (p=0.0007). Five BSI were associated with possible complications of HTLV-1; strongyloidiasis (3), infective dermatitis (1), HTLV-1 associated bronchiectasis (1). ConclusionsHigher HTLV-1c PVL predicts BSI risk; however; most BSI were not associated with recognised complications of HTLV-1 infection.

HTLV-1 drives vigorous clonal expansion of infected CD8(+) T cells in natural infection.

BackgroundHuman T-lymphotropic Virus Type I (HTLV-1) is a retrovirus that persistently infects 5–10 million individuals worldwide and causes disabling or fatal inflammatory and malignant diseases. The majority of the HTLV-1 proviral load is found in CD4+ T cells, and the phenotype of adult T cell leukemia (ATL) is typically CD4+. HTLV-1 also infects CD8+ cells in vivo, but the relative abundance and clonal composition of the two infected subpopulations have not been studied. We used a high-throughput DNA sequencing protocol to map and quantify HTLV-1 proviral integration sites in separated populations of CD4+ cells, CD8+ cells and unsorted peripheral blood mononuclear cells from 12 HTLV-1-infected individuals.ResultsWe show that the infected CD8+ cells constitute a median of 5 % of the HTLV-1 proviral load. However, HTLV-1-infected CD8+ clones undergo much greater oligoclonal proliferation than the infected CD4+ clones in infected individuals, regardless of disease manifestation. The CD8+ clones are over-represented among the most abundant clones in the blood and are redetected even after several years.ConclusionsWe conclude that although they make up only 5 % of the proviral load, the HTLV-1-infected CD8+ T-cells make a major impact on the clonal composition of HTLV-1-infected cells in the blood. The greater degree of oligoclonal expansion observed in the infected CD8+ T cells, contrasts with the CD4+ phenotype of ATL; cases of CD8+ adult T-cell leukaemia/lymphoma are rare. This work is consistent with growing evidence that oligoclonal expansion of HTLV-1-infected cells is not sufficient for malignant transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0221-1) contains supplementary material, which is available to authorized users.

In vitro basal T-cell proliferation and HTLV-1 proviral load among HTLV-1 subjects co-infected with Hepatitis C and/or HIV-1

Background HTLV-1 infection may is present among HCV or HIVinfected subjects and is associated with higher risk for HAM/TSP and other inflammatory diseases. Lymphocytes from about half of HTLV-1-infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Objective: Evaluate T-cell proliferation in vitro and HTLV-1 proviral load (PVL) among co-infected subjects.

Effect of pulsed methylprednisolone on disease severity, viral load and inflammation in patients with human T-lymphotropic virus type 1 associated myelopathy

The efficacy of treatments used for patients with HTLV-1 associated myelopathy/tropical spastic paraperesis (HAM/TSP) is uncertain although corticosteroids are widely prescribed. The effect of pulsed IV methylprednisolone was retrospectively analysed in an open cohort of 26 patients. 1g IV methylprednisolone was infused on three consecutive days. The outcomes were pain, gait, urinary frequency and nocturia, a range of inflammatory markers and HTLV-1 proviral load. A plasma cytokine profile was conducted in nine patients and correlated to gait and pain. Significant improvements in pain and 10m timed walk were observed immediately after the 3rd infusion and maintained, for pain, for up to six months. A shorter duration of disease was strongly correlated with improvement in the 10m timed walk (p=0.05) but not with the reduction in pain. Although baseline cytokine concentrations did not correlate to baseline pain or gait impairment a decrease in tumour necrosis factor-alpha (TNF-α) concentration after pulsed methylprednisolone was associated with improvements in both. Pulsed IV methylprednisolone significantly reduced pain in patients with HAM/TSP and was associated with a transient improvement in gait that may be related to a reduction in TNF-α concentration.

Droplet Digital PCR for absolute quantification and determination of proviral load of HTLV-1 and 2

HTLV proviral load in PBMCs is one of the few laboratory parameters used for clinical follow up and assessment of development of symptomatic disease associated with HTLV infection. High levels of proviral load are associated with symptomatic disease (e.g. ATLL, HAM/TSP) and occurance of higher levels among asymptomatic individuals has been suggested prognostic for development of symptomatic HTLV disease. Commonly, standard PCR methods including standard curves have been used. Droplet Digital PCR (ddPCR) is a new method that enables absolute quantification of DNA and RNA without the use of a standard curve. In ddPCR, the PCR sample is partitioned into 20 0 nanoliter-sized droplets prior to PCR and based on the number of positive droplets and Poisson algorithms the concentration of the sample can be determined. We have established and evaluated an assay for identification, quantification and determination of PVL of HTLV-1 and 2 in PBMC using ddPCR. For determination of PVL, the human gene RPP30 was quantified and used as reference [1]. The samples were extracted using the QIAamp DNA Blood Mini kit on the QIAcube (Qiagen) prior to ddPCR. Sixty HTLV-1 – positive (PVL 0.01%-59.8%) and 12 HTLV-2 – positive samples (PVL 0.02%-11.3%) previously analyzed with real-time PCR at Imperial College, St Mary's Hospital, London (Prof Graham Taylor) as well as 20 negative samples and dilution series of both HTLV-1 and 2 were examined. The results of ddPCR were in concordance with real-time-PCR, however a few weak positive samples were only detected by one of the methods, see Figure 1. The detection levels of the ddPCR were determined to 1-2 copies per PCR reaction for both HTLV-1 and 2. Both the intra- and inter-assay variability of ddPCR was low, 0.97-3.34% and 1.84-6.13% respectively. In conclusion, ddPCR allows for reliable and sensitive determination of PVL for both HTLV-1 and 2.

Deep sequencing of the TCRβ repertoire reveals T cell clonal expansion is associated with HTLV-1 proviral load in HAM/TSP patients

HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with a chronic inflammatory central nervous system in which the host immune response against HTLV-1 infection is considered immunopathogenic. We have applied a new unbiased deep sequencing method of TCR repertoire to accurately measure the diversity and degree of clonal expansion of peripheral circulating T-cells in patients with HAM/TSP compared to age, gender, and ethnicity matched healthy controls. TCR libraries were generated using 5'rapid amplification of cDNA ends to amplify the V-D-J genes of unsorted T-cells obtained from peripheral blood. Over 100 million reads were analyzed using MiGec software (Shugay, et al., Nat Methods. 2014 Jun;11(6):653-5), that align and match the human TCR β-chain nucleotide sequences through IMGT database. T-cell clonotypes with high-quality CDR3 sequence were selected by a molecular identifier PCR strategy, which allowed for filtering PCR errors and correct for artificial sequences generated on a HiSeq 2000 Illumina system platform. By calculating the coefficient of variation of our dataset we considered clonal expansion as those unique TCR clonotypes showing reads >8. While we did not observe any significant statistical differences of the number of single clones (clonotypes with 1 read, P=0.62) between HAM/TSP patients and healthy controls groups, by contrast, a higher clonal expansion of the T-cell repertoire was observed in HAM/TSP patients when compared to healthy controls (unpaired parametric T-test with P=0.026). Interestingly, this clonal expansion of the HAM/TSP TCR repertoire correlated with the HTLV-I provirus load (as defined by digital PCR quantification of the HTLV-I tax gene) (r=0.64, P=0.008). Our findings strongly suggest that in this chronic neurological disorder, a strong dysregulated T-cell response may be driven by HTLV-I and that strategies to decrease virus may be of clinical benefit.

Immunophenotyping of cerebrospinal fluid cells in virus-associated neurologic diseases

OBJECTIVE: To characterize the cellular immunophenotype of cells in cerebrospinal fluid (CSF) of patients with virus-associated neurologic diseases. BACKGROUND: Various inflammatory neurologic diseases are associated with viral infections. These agents may cause direct damage of infected cells associated with immunological alterations such as chronic activation, immunodeficiency and infiltration of inflammatory cells into the CNS that underlie the pathogenesis of inflammatory neurologic diseases. Therefore, characterization of local immune responses that are associated with the inflammatory milieu may provide evidence or pathogenic “signature” of an immunopathogenic process in virus-associated neurologic diseases. DESIGN/METHODS: Multicolor flow cytometry is used to characterize CD4+ and CD8+ T cells, B cells, monocytes and NK cells in the CSF and blood of patients with virus-associated neurologic diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP; n=16), HIV adequately treated with antiretroviral drugs (n=16), multiple sclerosis (MS; n=4) and progressive multifocal leukoencephalopathy (PML; n=6) compared to healthy volunteers (n=6). RESULTS: In all patients, T cells were the predominant population in the CSF. CD4+ and CD8+ T cells were significantly altered among the groups, and the CD4/CD8 ratio reduced in HIV, HAM/TSP and a subset of PML, but elevated in MS. In patients with HAM/TSP, activated CD4+ T cells were significantly increased in the CSF as well as the blood and correlated with HTLV-1 proviral DNA loads. MS patients had a decreased frequency of effector/memory CD4+ and CD8+ T cells in the CSF. B cells and antibody producing plasma cells were elevated in patients with HAM/TSP, MS and PML whereas monocytes were reduced. CD56bright/CD56dim NK cell ratio was elevated in the CSF of patients with HAM/TSP. CONCLUSIONS: Immunophenotyping of CSF cells may reflect immune pathology in inflammatory neurologic diseases and can serve to highlight differential diagnoses that may lead to a better understanding of disease pathogenesis and clinical treatment. Disclosure: Dr. Akahata has nothing to disclose. Dr. Massoud has nothing to disclose. Dr. Caruso has nothing to disclose. Dr. Ohayon has nothing to disclose. Dr. Billioux has nothing to disclose. Dr. Von Geldern has nothing to disclose. Dr. Smith has nothing to disclose. Dr. Nath has nothing to disclose. Dr. Jacobson has nothing to disclose.

Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via de novo infection (infectious spread) and infected cell proliferation (mitotic spread), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). However, the relative contributions of these mechanisms are unknown, and will determine the efficacy of antiretroviral therapy. The prevailing view is that infectious spread does not contribute to HTLV-1 proviral load after early infection. However, this notion is based on low-throughput and imprecise data on clonal integration site abundance and diversity. We recently used our high-throughput protocol that maps and quantifies the abundance of HTLV-1 integration sites to estimate a diversity of between 10^4 and 10^5 HTLV-1 T cell clones in the body of an asymptomatic carrier or patient with HAM/TSP. Since infectious spread establishes new clones, high clonal diversity implies substantial infectious spread. HTLV-1 clonal abundance varies within individual hosts by several orders of magnitude. Therefore quantifying within-host HTLV-1 dynamics requires mathematical modelling at multiple scales, and stochastic processes are necessary to describe the behaviour of small clones. Here we apply a hybrid model of deterministic and stochastic processes to time-series patient data, to estimate the relative contributions of infectious spread and mitotic spread. We find, contrary to previous belief that infectious spread persists during chronic infection, even after the proviral load has reached its set point. The risk of HTLV-1-associated malignant and inflammatory disease is strongly correlated with the proviral load: the load in turn is correlated with the total number of HTLV-1-infected clones, but not with the degree of oligoclonal proliferation. Our results therefore suggest that attempts to suppress de novo infection may reduce the risk of malignant transformation.

Humanized anti CCR4 antibody KW0761 targets HTLV-1 infected CD4+ CCR4+ and CD8+CCR4+ T cells to treat HAM/TSP

Human T-lymphotropic virus type I (HTLV-1) can cause HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma (ATL). Since the prognosis for HAM/TSP patients is extremely poor, there is a strong demand for a novel therapeutic strategy, especially one which would effectively reduce HTLV-1 proviral load, which is well-correlated with disease prognosis. CD4+CCR4+ T cells are the main HTLV-1 reservoir, and the defucosylated humanized anti-CCR4 antibody KW0761 has been approved in Japan as a treatment for ATL. KW0761 strongly binds to Fcγ receptor IIIa (FcγRIIIa) on natural killer cells and elicits powerful antibody-dependent cellular cytotoxicity (ADCC) against the CCR4+ cells. In this study, we evaluated KW0761 as a treatment for HAM/TSP using primarily ex vivo cell cultures. In addition, given that KW0761 would also target CD8+CCR4+ T cells, we sought to determine how this would likely affect HAM/TSP patients by elucidating the role of CD8+CCR4+ T cells in HAM/TSP pathogenesis. When applied to cultures of PBMCs from HAM/TSP patients (n=11), KW0761 effectively reduced HTLV-1 proviral load (56.4% mean reduction at 0.01μg/mL), spontaneous proliferation, and production of pro-inflammatory cytokines including IFN-γ. Like CD4+CCR4+ T cells, CD8+CCR4+ T cells from HAM/TSP patients exhibited high proviral loads and spontaneous IFN-γ production, unlike their CCR4-counterparts. CD8+CCR4+ T cells from HAM/TSP patients contained more IFN-γ+ cells and less IL-4+ cells than those from healthy donors. Notably, Tax-specific cytotoxic T lymphocytes that may help control the HTLV-1 infection were overwhelmingly CCR4-. In conclusion, we determined that CD8+CCR4+ T cells as well as CD4+CCR4+ T cells are prime therapeutic targets for treating HAM/TSP, and that KW0761 shows promise as a new treatment. Based on the results of this study, we have begun conducting an investigator -led Phase I/IIa clinical trial to test the safety and efficacy of KW0761 on HAM/TSP patients (UMI12655).

DARC polymorphism that abrogates erythrocyte Duffy antigen expression influences MCP-1 plasma level in HTLV-1 infected individuals but are not associated with HAM/TSP

Genetic host factors influence the outcome of HTLV-1 infection. Chemokines are important in the immune response against virus, and play a role in HAM/TSP pathogenesis. Duffy antigen receptor for chemokines (DARC) functions as a chemokine reservoir, and DARC polymorphisms rs12075 (A>G; FY*B>FY*A) and rs281477 (-46T>C; FY*B>FY*BES) may influence circulating concentrations of proinflammatory chemokines. We investigate whether Duffy genotypes influence HTLV-1 proviral load (pvl) level and plasma chemokines concentrations. HTLV-1 asymptomatic carries (AC=162), HAM/TSP patients (HAM=146) and seronegative individuals (SN=71) were genotyped for FY*A, FY*B and FY*BES. Quantification of plasmatic IL-8, MCP-1 and RANTES were performed by flow cytometry for all participants. HTLV-1 pvl was quantified in peripheral blood. The frequency of Duffy haplotypes was not significantly different among the three groups. Comparison of individuals with different Duffy haplotypes showed that pvl was significantly higher in HAM than in AC group, but not showed differences intragroup. IL-8 level was significantly higher in HAM than in AC and SN, and was higher in AC than in SN. Otherwise, MCP-1 was significantly higher in SN than in AC and HAM, but was not different when comparing AC and HAM. The highest RANTES level was seen in SN group, and the difference between SN and AC was significant, but not between SN and HAM, or between infected groups. Surprisingly, in a recessive genetic model, corrected by age and gender, the polymorphism -46C/C that abrogates erythrocyte Duffy antigen expression was significantly associated with lower level of MCP-1 in AC group, in HAM group and in all infected individuals, but not in SN group. We conclude that the Duffy null genotype (-46C/C) diminishes the plasma concentration of MCP-1 in HTLV-1 infected individuals, but this influence does not appear to be associated with HAM/TSP development. Financial support: Fundacao Hemominas and FAPEMIG.

Are seroindeterminate western blot patterns in human T-Cell lymphotropic virus type 1 (Htlv-1) infected individuals associated with low proviral load levels?

For HTLV-1/2 diagnosis, reactive screening results are generally confirmed by Western Blot (WB). However, the large number of indeterminate WB patterns is still a problem worldwide. The aim of this study was to determine whether low levels of proviral load (PVL) are associated with seroindeterminate results by WB. PVL was determined in 49 HTLV-1 samples confirmed by n-PCR and classified as G1: positive by WB from individuals with disease (n= 27, 5 ATLL and 22 HAM/TSP); G2: positive by WB from asymptomatic carriers (n= 18); and G3: seroindeterminate samples by WB from asymptomatic carriers (n= 4). The viral gen pol and albumin were quantified by real-time SYBR Green PCR (ABI Prism System-AppliedBiosystems 75). Calibration curves were constructed using a DNA stock from MT2 cell line (Limit of quantification: 3 pol copies/reaction-R2> 0.99) and the analysis was performed by Krustall Wallis (GraphPad Prism v.5). Median PVL values were 4.03, 1.58 and 0.15 copies of HTLV-1/1 PBMCs fo r G1, G2 and G3, respectively. Correlation between PVL and age of patients (S= 0.61) was found for G1 samples. PVL median values were significantly different between the three groups (p= 0.3); the difference was also observed (p = 0.5) when considering HTLV-1 positive samples by WB [G1 + G2] as a single group. Even though a seroconversion could not be discarded in seroindeterminate cases, a low viral replication rate due to other factors could trigger a weak immune response, thus causing seroindeterminate WB patterns. The present study clearly demonstrates that such cases could be associated to a low HTLV-1 PVL and that the molecular analysis is a necessary tool to reach a final diagnosis.

HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

Plasma of patients infected with HTLV-1 is considered non-infectious but detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential value in diagnosis and prognosis. RNA from 1 ml of plasma from 65 subjects infected with HTLV-1 (27 asymptomatic carriers [AC]), 17 patients with HTLV-1-associated myelopathy (HAM/TSP), 14 with adult T-cell leukemia/lymphoma (ATLL), two co-infected with HIV, and five with other HTLV-1-associated disease, was extracted and reverse transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis. HTLV-1 RNA was detected in plasma from 18 (28%) patients, always at a very low copy number (3-13 copies viral cDNA per milliliter of plasma). Mean values of HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma. HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor.

HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis Is Not Associated with SNP rs12979860 of the IL-28B Gene.

The present study investigated the association between the rs12979860 polymorphism in the IL-28B gene and HTLV-1 infection as well as the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected patients (26 HAM/TSP symptomatic and 53 asymptomatic) and 300 seronegative healthy controls were investigated. Plasma levels of the cytokines TNF-α, TNF-β, IL-8, IL-10, IL-6, and IFN-γ from infected patients were measured using an indirect enzyme-linked immunosorbent assay. The HTLV proviral load was measured using a real-time PCR assay, and T-cell subset counts were determined by flow cytometry. Real-time PCR was used to genotype the rs12979860 SNP. The allelic and genotypic distributions displayed no significant differences among the investigated groups. No significant association between the serum cytokine levels and the presence of the rs12979860 SNP in symptomatic and asymptomatic subjects was observed. A positive correlation (p = 0.0015) between TNF-β and IFN-γ was observed in the asymptomatic group, but a positive correlation was only observed (p = 0.0180) between TNF-α and IL-6 in the HAM/TSP group. The proviral load was significantly higher in HAM/TSP patients than in asymptomatic subjects. The present results do not support a previous report indicating an association between the SNP rs12979860 and HAM/TSP outcome.

Functional Activity of Monocytes and Macrophages in HTLV-1 Infected Subjects

The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing inflammatory activity of macrophages was similar in HAM/TSP patients and HC and it was related to HTLV-1 proviral load rather than the high IFN-γ production observed in these subjects.

Achievement of Complete Hematological Remission and Reversal of High HTLV-1 Viral Loads with Anti CCR-4 Monoclonal Antibody Administration in Afro Caribbean Patients with Relapsed Chronic Adult T Cell Leukaemia (ATLL)

IntroductionThe management of heavily pre-treated patients with relapsed ATLL is challenging. These patients display chemo-refractory disease with limited therapeutic options. The Kyowa Hakko Kirin protocol 0761-009 is a randomized controlled trial of anti-CCR4 monoclonal antibody therapy (mogamulizumab) compared with physician choice of salvage chemotherapy in relapsed ATLL. ATLL arises in HTLV-1 infected patients with high pre-morbid HTLV-1 proviral loads (>4–10 HTLV-1 DNA copies/ml)1.MethodsThree patients, female, age 38 - 72 years and of Afro Caribbean origin, with relapsed chronic ATLL, have been recruited to 0761-009 in London. All patients had received two or more lines of therapy: #1 zidovudine (ZDV)/Interferon-a (IFN), anti-CD25/bortezomib; #2 ZDV/INF, anti-CD25/bortezomib, etoposide, Gemcitabine/Oxaloplatin; #3 sodium valproate, ZDV/IFN, etoposide. Lymphocyte counts at study entry were 7.8 - 33.5 x 109/L with high HTLV-1 proviral loads (range 25.4 - 122.4 HTLV-1 DNA copies/100 PBMC). Anti-CCR4 mAb was administered at a dose of 1mg/kg over 1 hour, weekly for 4 weeks and every 2 weeks thereafter. Data were censored 17/06/2014.ResultsAll three patients achieved complete hematological remission (CR) after 1 – 2 infusions. Patient #1 remains on treatment and in CR day 397. Patient #2 received 3 treatment cycles with normalization of lymphocyte count and complete resolution of extensive cutaneous lesions (baseline SWAT= 93.6) but treatment was discontinued after 3 cycles due to, now resolved, drug toxicity. Patient #3 remains on treatment and in CR at day 139. In each case HTLV proviral load fell to 0.02% < 0.7% and have remained <1% for the duration of therapy.ConclusionsAlthough a small case series these data illustrate the efficacy of anti-CCR4 mAb in ATLL, particularly in the chronic form, despite prior treatment failure and support the data observed in the Japanese cohort where CR was achieved in patients with blood compartmental disease, particularly chronic ATLL2. However, the extent of reduction in HTLV-1 proviral load with anti-CCR4 was not observed previously in these patients (or others who achieved CR with zidovudine/interferon the first line treatment of chronic ATLL). The reduction in HTLV-1 proviral burden to <1% suggests not only clearance of circulating HTLV-1 infected malignant clones but additional depletion of HTLV-1 infected non-malignant clones. This will be confirmed by unique integration site analysis.Reference List(1) Hodson A, Laydon D, Bain BJ, Fields P, Taylor GP. Pre-morbid HTLV-1 proviral load, rather than percentage of abnormal lymphocytes, is associated with an increased risk of aggressive ATLL. Haematologica2013; 98(3):385-388.(2) Ishida T, Joh T, Uike N, Yamamoto K, Utsunomiya A, Yoshida S et al. Defucosylated anti-CCR4 monoclonal antibody (KW-0761) for relapsed adult T-cell leukemia-lymphoma: a multicenter phase II study. J Clin Oncol 2012; 30(8):837-842. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Peripheral Blood Stem Cell Transplantation Using Reduced-Intensity Conditioning Regimen with Fludarabine and Busulfan from HLA-Matched Related Donor for Elderly Adult T-Cell Leukemia/Lymphoma: Results of Multicenter Phase II Study (ATL-NST-3)

Background: Adult T-cell leukemia-lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection and commonly affects individuals at an average age of 67 years. Since the prognosis of aggressive ATL patients (pts) treated with conventional chemotherapy has been dismal, we had so far conducted two clinical trials to evaluate the feasibility of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an HLA-identical sibling donor using reduced-intensity conditioning regimen (RIC) consisting of fludarabine and busulfan. We reported that allo-PBSCT using RIC provided a long-term survival in about one third of ATL pts and that it exerted not only graft-versus-ATL effects but also graft-versus-HTLV-1 effects (Okamura J, et al. Blood 2005; Tanosaki R, et al. Biol Blood Marrow Transplant 2008; Choi IL, Bone Marrow Transplant 2011). To confirm these promising results in a larger number of ATL pts, we conducted a phase II study (UMIN C000000409).Patients and Methods: Between September 2006 and July 2011, 20 pts were registered in this study. Because ATL is a rare disease affecting elderly people, their siblings were also old and were not appropriate for donors, and the enrollment of pts needed much more time than we had expected. Therefore, though we had originally planned to enroll 35 pts, we had to close this study incompletely. The conditioning regimen consisted of fludarabine 30 mg/m2/day intravenously days -8 to -3, and busulfan 3.2 mg/kg/day intravenously or 4 mg/kg/day orally days -6 and -5. GVHD prophylaxis was CsA alone from day -1 and PBSCs (CD34+ cells >2x106/kg) were infused on day 0. Primary endpoint was 2-year overall survival (OS). Engraftment, adverse events such as graft-versus-host disease (GVHD) and infectious events, and progression free survival (PFS) were also evaluated as well as donor chimerism by STR-PCR analysis, HTLV-1 proviral load, and Tax-specific cytotoxic T lymphocytes (CTL) by tetramers. The eligibility criteria at registration included acute- or lymphoma-type ATL, pts with an HLA-matched related donor, age between 50 and 70, disease status of CR, PR or NC with stable disease, no uncontrollable active infections, no major organ dysfunction, and no active CNS involvement.Results: Median age was 54 years (range 50-68), genders were 10 males and 10 females, ATL types were 13 acute and 7 lymphoma, and disease status at transplantation was 9 CR, 8 PR, and 3 NC. 13 donors were HTLV-1 positive. Engraftment as confirmed by donor chimerism analysis was achieved in all pts. OS and PFS were 95% (95%CI, 86-100%) and 60% (95%CI, 39-82%) at day 100, 74% (95%CI, 55-94%) and 45% (95%CI, 23-67%) at 1 year, and 53% (95%CI, 31-76%) and 40% (95%CI, 19-62%) at 2 years. Acute GVHD occurred in 13 pts (grade I in 2, II in 5, III in 6 pts), and chronic GVHD developed in 12 pts (63%) out of 19 evaluable pts. Eleven pts died at the time of analysis; ATL 6, GVHD 3, IP 1, PCP 1. There was no significant relationship between OS and grades of acute GVHD. While HTLV-1 proviral load decreased as early as 1 month after transplantation and became undetectable level in 14 pts (70%) out of 20, the increase of A2- or A24-restricted Tax-specific CTLs in HLA-A2 or A24-positive pts, respectively, were observed in some pts after 3 months or later, and there was no clear relationship between them. With a median follow-up of 36 months (range 24-86), 9 pts (5 CR and 4 PR at transplantation) were alive without disease.Conclusions: It was confirmed that allo-PBSCT using RIC consisting of fludarabine and busulfan was relatively safe and effective, and provided a long-term survival at least in chemo-sensitive aggressive ATL pts. DisclosuresNakamae:Novartis: Honoraria, Research Funding, Speakers Bureau, Travel/accomidations/meeting expenses Other.