HTLV-1 Infected Individuals Research Articles

Mannose binding lectin-associated serine protease 2 (MASP2) gene polymorphism and susceptibility to human T-lymphotropic virus type 1 (HTLV-1) infection in blood donors from Mashhad, Iran.

Mannose binding lectin-associated serine protease 2 (MASP2) is the effector part of mannose binding lectin (MBL) that activates the complement system in an antibody-independent manner. We aimed to investigate the role of genetic polymorphisms in the MASP2 gene and susceptibility to HTLV-1 infection. A total of 172 HTLV-1 infected individuals and 170 healthy blood donors were analyzed in this case-control study. Nine single nucleotide polymorphisms (SNPs) encompassing different regions of the MASP2 gene were genotyped with a polymerase chain reaction-sequence-specific primer (PCR-SSP) assay. The relation between the SNPs genotype and the susceptibility to HTLV-1 infection was investigated with a χ2 test considering P < 0.05 as statistically significant. Two of nine tested SNPs were associated with the risk of HTLV-1 infection. The genotype TT at rs17409276 decreased the risk of HTLV-1 (P = 0.005, OR = 0.301, 95% CI = 0.124-0.728). The genotypes CC and CT at rs2273346 were also associated with a higher risk of HTLV-1 acquisition (P = 0.004, OR = 2.225, 95% CI = 1.277-3.877). These findings highlight the importance of MASP2 genetic polymorphisms in the lectin pathway of complement activation and susceptibility to HTLV-1 infection.

EOS, an Ikaros family zinc finger transcription factor, interacts with the HTLV-1 oncoprotein Tax and is downregulated in peripheral blood mononuclear cells of HTLV-1-infected individuals, irrespective of clinical statuses

BackgroundEOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).MethodsIn this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated.ResultsEOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ. Meanwhile, there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses. We found an inverse correlation between EOS mRNA levels and HTLV-1 PVL in ATL patients, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP patients, whereas this correlation was not observed in other clinical statuses.ConclusionsThese findings suggest that both Tax and HBZ can alter the expression of EOS through undetermined mechanisms, and dysregulated expression of EOS in PBMCs of HTLV-1 infected individuals may contribute to the pathological progression of HTLV-1-associated diseases, such as ATL and HAM/TSP.

Functional capacity of natural killer cells in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

BackgroundNatural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection.MethodsThis cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30.ResultsThe frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30–61] compared to controls (73%, IQR 54–79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals.ConclusionsNK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.

Lack of association between single-nucleotide polymorphisms of pro- and anti-inflammatory cytokines and HTLV-1-associated myelopathy / tropical spastic paraparesis development in patients from Rio de Janeiro, Brazil

BackgroundHTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive neurological and inflammatory disease, associated with HTLV-1 infection. HAM/TSP neurological disease is a consequence of an inflammatory reaction, and adaptive immune responses, through the secretion of anti-inflammatory and pro-inflammatory cytokines, play an important role in the outcome of infection and disease progression. Studies addressing the association between cytokines functional single nucleotide polymorphisms and HAM/TSP development are scarce.MethodsThe genetic polymorphisms of cytokine genes were evaluated in HAM/TSP patients (n = 68) and in asymptomatic HTLV-1 positive carriers (n = 83) from Rio de Janeiro, Brazil, in a case-control study. HTLV-1 infected patients were genotyped for SNPs in five cytokine genes: TNFA-308G/A, IL6-174G/C, IFNG + 874 T/A, TGFB at the codons + 10 T/C and + 25G/C, IL10-592C/A and -819C/T, and -1082A/G and proviral load (PVL) was quantified. Associations between genotypes, haplotypes, clinical outcome and pro viral load were evaluated.ResultsLack of association between the cytokine polymorphisms and disease outcome was observed. The genotypes TNFA-308GG, IL6-174GG/GC, IL10-592AA and -819CC and TGFb1 high producers phenotypes were correlated with higher PVL in HAM/TSP patients versus asymptomatic carriers.ConclusionsWe did not observe association between cytokine polymorphisms and risk for HAM/TSP development in Brazilian HTLV-1 infected individuals, regardless of differences in PVL between HAM/TSP versus asymptomatic carriers in specific cytokine polymorphisms.

Estimation of HTLV-1 vertical transmission cases in Brazil per annum.

BackgroundBrazil has at least 800,000 HTLV-1 infected individuals. HTLV-1 can be transmitted via sexual intercourse, contact with blood and from mother to child, mainly by breastfeeding. Treatments for the high morbidity/mortality associated diseases (ATL and HAM/TSP) are limited, therefore, infection prevention is of utmost importance. However, antenatal screening is not routinely performed in Brazil. A lack of data regarding the number of individuals infected via breastfeeding impairs the development of government policies. The objective is to estimate the number of HTLV-1 infections occurring annually due to mother to child transmission (MTCT) in Brazil, nationally and regionally.MethodologyTo estimate HTLV-1 MTCT in Brazil the following variables are modelled: number of births, prevalence of HTLV-1 infection in pregnant women, breastfeeding duration rate and transmission risk according to breastfeeding period. The number of cases of HAM/TSP and ATL attributable to MTCT are also estimated.Principal findingsIn 2008, there were 2,934,828 live births in Brazil. HTLV prevalence in pregnant women in Brazil ranges between 0.1–1.05% by region. An estimated 16,548 HTLV-1 infected women are pregnant each year. According to the breastfeeding pattern and HTLV-1 prevalence of each region there are an estimated 3,024 new cases of HTLV-1 infection due to MTCT annually of which 2,610 are preventable through infant feeding advice. These 3,024 transmissions will result in 120–604 cases of ATL and 8–272 of HAM/TSP. North-East region comprises the high number of MTCT cases, followed by South-East.Conclusions/significanceA high number of new HTLV-1 infections due to MTCT occur every year in Brazil. Antenatal screening and avoiding breastfeeding are essential to prevent subsequent development of HTLV-1-associated diseases.

Local and systemic production of proinflammatory chemokines in the pathogenesis of HAM/TSP

BackgroundHTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is related with high proviral load, high proinflammatory cytokine levels, and passage of infected cell from the blood to the central nervous system. We aimed to evaluate the participation of chemokines and adhesion molecules in HAM/TSP pathogenesis. MethodsCXCL9, CXCL10, sICAM-1, and sVCAM-1 were determined by ELISA in serum and cerebrospinal fluid (CSF) of HTLV-1 infected individuals. The frequency and median fluorescence intensity (MFI) of lymphocytes and monocytes expressing ligands of adhesion molecules (CD11a and CD49d) and a chemokine receptor (CXCR3) were analyzed by flow cytometry. ResultsThe levels of CXCL9 and CXCL10 in serum of definite HAM/TSP were higher than in serum of probable HAM/TSP and HTLV-1 carriers. Considering the production of chemokines by patients with definite HAM/TSP, CXCL9 levels were higher in serum than in CSF, and CXCL10 production was higher in CSF than in serum. Levels of adhesion molecules in serum and CSF of HTLV-1 infected individuals did not differ. The MFI of CD11a on CD4+, CD8+ and CD14+ cells was lower in definite HAM/TSP than in HTLV-1 carriers and did not differ from probable HAM/TSP and healthy subjects (HS). The frequency of lymphocytes expressing CXCR3 was lower in definite HAM/TSP than in cells of probable HAM/TSP and did not differ from carrier and HS. ConclusionThese data point to the participation of proinflammatory chemokines, especially CXCL10, in the pathogenesis of definite HAM/TSP.

Plasma and cerebrospinal fluid levels of cytokines as disease markers of neurologic manifestation in long-term HTLV-1 infected individuals.

The aim of this study is to evaluate the presence of a particular immunological profile in individuals long-term infected with HTLV-1, followed presenting different clinical courses. Forty-eight individuals were evaluated for 19 cytokines analyzed in cerebrospinal fluid and plasma of patients with HTLV-1 presenting with and without neurological symptoms. Proinflammatory cytokines and the chemokine ligand 11 (ITAC/CXCL11) were increased in individuals with HTLV-1 coursing with neurological symptoms. Different cytokines' expression profile in the presence of neurological symptoms may help to understand and characterize the progression for severe clinical presentations.

Detection of HTLV-1 proviral DNA in BM mononuclear cells and cultured mesenchymal stromal cells isolated from patients with HTLV-1 infection

The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4+ T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4+ T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4+ T-lymphocytes infiltrated into the bone marrow.

Impaired TNF, IL-1β, and IL-17 production and increased susceptibility to Mycobacterium tuberculosis infection in HTLV-1 infected individuals

IFN-γ and TNF play critical roles in the control of Mycobacterium tuberculosis infection. Despite leading to an exaggerated production of inflammatory cytokines, HTLV-1 infection increases the risk of developing tuberculosis (TB). However, the immune mechanisms accounting for this phenomenon are still unclear. The aim of this study was to evaluate immunological aspects of the HTLV-1/M. tuberculosis co-infection. In this cross-sectional study, the levels of TNF, IL-1β, and IL-17 were determined by ELISA in the supernatants of either unstimulated or tuberculin purified protein derivative (PPD) stimulated peripheral blood mononuclear cells. Cells from HTLV-1 infected individuals produced lower levels of TNF following PPD stimulation compared to unstimulated cells. IL-1β and IL-17 production by cells from HTLV-1/M. tuberculosis co-infected individuals was lower than in cells from patients with TB. Impairment in TNF, IL-1β, and IL-17 production upon stimulation with mycobacterial antigens may contribute to the increased susceptibility to M. tuberculosis infection observed in HTLV-1 infected individuals.

HTLV-1 is predominantly sexually transmitted in Salvador, the city with the highest HTLV-1 prevalence in Brazil.

BackgroundSalvador is the city with the highest number of HTLV-1 infected individuals in Brazil, yet the main route of HTLV-1 transmission is unknown.ObjectiveTo investigate the association of syphilis infection as a proxy for sexual transmission of HTLV-1 infection in the general population of this city.MethodsA cross sectional population-based study was conducted with 3,451 serum samples obtained by a representative simple random sampling. Data on gender, age, income, and years of education were collected by questionnaire and the presence of HTLV, HIV and Treponema pallidum infection was determined by serology. Logistic regression analysis was used to evaluate the independent effect of the potential explanatory variables to HTLV-1 infection and Odds Ratios (OR) and 95% CI were calculated.ResultsThe majority of studied individuals were female (56.4%), had less than 7 years of education (55.3%) and earned two or less minimum wages (52.0%). The overall prevalence of HTLV-1 was 1.48% (51/3,451; 95% CI: 1.10%– 1.94%), which increased with age. Only three persons younger than 17 (3/958; 0.31%; CI 95% 0.06–0.91) years were infected by HTLV-1. Among the 45 syphilis positives, 12 (26.7%) were HTLV positive, while among 21 HIV positives, only one (4.8%) was HTLV positive. HTLV-1 infection was found to be associated with syphilis infection (ORADJUSTED 36.77; 95% CI 14.96–90.41).ConclusionThe data presented herein indicate that horizontal transmission between adults is the main route of HTLV-1 infection in the general population of Salvador and that this is likely to occur through sexual contact.

P-E3 Development of a new humanized mouse model to study anti-HTLV-1 immunity

HTLV-1 is the only known human virus that causes leukemia (adult-T cell leukemia, ATL), which is a fatal disorder without established treatment at the moment. Thus, the development of a new treatment represents an unmet medical need. In collaboration with the stem-cell transplant group of the Cancer Center of the University of Maryland, we have conducted a longitudinal study of ATL patients who have received allogeneic stem-cell transplant (AlloSCT) and observed that the restoration of competent anti-HTLV-1 T-cell immunity coincided with the suppression of ATL relapse in these patients. On the other hands, the ATL patients in their acute leukemic phase showed almost negligibleanti-HTLV-1 immunity, whereas HTLV-1 infected individuals in their asymptomatic latency phase show detectable immunity against this virus. These observation prompted us to hypothesize that the diminished T-cell immunity against HTLV-1 may underlie the transition from asymptomatic latency to acute leukemic phase. In particular, we observed that the anti-HTLV-1 CD8 T cells in acute ATL patients show signs of severe T-cell exhaustion. So we set a goal to test if reversion of T-cell exhaustion may help restore effective anti-HTLV-1 immunity and thereby allow to control the malignant expansion of HTLV-1+ CD4 cells in vivo. To test this hypothesis, we transferred the leukemic T-cells and non-leukemic lymphocytes from a few patients into severely immune compromised NSG (NOD-SCID/gc knockout) mice and observed the successful expansion of ATL leukemia and non-leukemic T and B cells from patients in these mice. Preliminary results suggest that anti-PD1 intervention augmented the homeostatic expansion of anti-HTLV-1 CD8 T cells in mice and contributed to suppressed proliferation of HTLV-1+ CD4 T cells in these mice. These results collectively suggest that we can develop a new anti-ATL strategy involving the anti-T-cell exhaustion to stop disease progression at the chronic phase, before the fatal acute expansion starts.

Polycomb-Dependent Epigenetic Landscape in Adult T Cell Leukemia (ATL); Providing Proof of Concept for Targeting EZH1/2 to Selectively Eliminate the HTLV-1 Infected Population

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma and is refractory to currently available combination chemotherapy. The unfavorable prognosis results from an inadequate understanding of how diseases are caused and maintained in human T-cell leukemia virus type I (HTLV-1)-infected individuals. To date, direct comprehensive analyses of leukemic cells have identified the intrinsic molecular hallmarks of ATL. Among these, polycomb group (PcG)-mediated epigenetic disruption has been known to be a crucial characteristic of ATL (Yamagishi et al., Cancer Cell, 2012). However, no attempt has been made to determine the global epigenomic status explaining the deregulated gene expression pattern specific to ATL.In this study, we performed integrative analyses of epigenome (n=3) and transcriptome (n=58) of primary ATL patient cells and corresponding normal CD4+ T cells to decipher the ATL-specific 'epigenetic-code' that was critical for cell identity. We found that PcG-mediated tri-methylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at over half of genes (53.8%) in ATL cells, the pattern of which appears distinct from other cancer types and PcG-dependent cell lineages such as ES cells and peripheral T lymphocytes. Large proportion of the abnormal gene downregulation occurred at an early stage of disease progression and was explained by the H3K27me3 accumulation. The global H3K27me3 alterations were involved in determination of key genes such as miR-31, CADM1, EVC1/2, CDKN1A, and NDRG2, which are essential for ATL cell survival and other cellular characteristics. In addition, PcG generated diverse outcomes by the remote regulation of a broad spectrum of gene regulators, including various transcription factors, miRNAs, epigenetic modifiers, and developmental genes. Thus, the emerging epigenomic landscape is a fundamental characteristic of ATL.Although EZH2 mutations conferring gain-of-function were undetected in ATL (0/50; 0%), EZH2 level was significantly upregulated and inversely correlated with H3K27me3 targets, indicating that the global alteration of H3K27me3 mark depends on the abundance of EZH2 and other core components of the polycomb repressive complex 2 (PRC2). We found that EZH2 was sensitive to promiscuous signaling network including NF-kB pathway and was functionally affected by HTLV-1 Tax through both NF-kB activation and direct association. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, the majority of epigenetic silencing has occurred in leukemic cells from indolent type ATL and also in HTLV-1-infected T cells from asymptomatic HTLV-1 carriers. Collectively, our results unveiled that epigenetic reprogramming arises at an early stage of ATL development.Tracing the epigenetic marks and expression patterns in samples obtained from patients of various diagnostic categories, as well as in other biologically relevant models has supported the efficacy and relevance of targeting PRC2. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, permitting favorable treatments. According to the expression profiling, EZH2 may represent the first-choice as a druggable target. In addition, peripheral T cells highly express EZH1 that compensates for the EZH2 functions. We found that simultaneous depletion of the two H3K27me3 writers EZH1 and EZH2 significantly diminished cellular H3K27me3 level and dramatically inhibited ATL cell growth compared with single depletion, suggesting that the compensatory actions of EZH1/2 may be critical for ATL.To selectively eliminate the ATL and HTLV-1-infetced populations, we have developed a novel EZH1/2 dual-inhibitor that shows strong inhibitory effects for both of EZH1 and EZH2. Treatment with the new drugs showed significant inhibitory effects on the ATL cell survival derived from patients. Interestingly, the dual-inhibitor reversed the epigenetic disruption and selectively eliminated leukemic and immortalized cells from HTLV-1 infected individuals. Collectively, this approach will achieve potent and selective synthetic lethality by targeting the regulators of H3K27me3 in aggressive and indolent ATL cells, as well as in clonally expanded infected cells, improving medical care and the prevention of disease onset. DisclosuresYamagishi:Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

HTLV-1 burden dependent on hijacking monocytes chemotaxis

The HTLV-DNA burden in PBMCs is a risk factor for HAM/TSP and ATL development. We investigated the contribution of monocyte subsets (classical, intermediate and non-classical) to the total viral burden in 23 HTLV-1 infected individuals by assessing their frequency, their chemotactic and phagocytic functions, as well as their infectivity status. Classical monocytes differed between infected and uninfected individuals, their frequency was lower and their expression level of the chemokine receptors CCR5, CXCR3 and CX3CR1 was higher. While the percentage and surface chemokine receptor expression did not differ between HTLV-1 infected and uninfected individuals, intermediate monocytes from HTLV-1 infected individuals had increased migratory capacity to CCL5, the ligand for CCR5. Non-classical monocytes from HTLV-1 infected individuals increased in frequency and expressed high levels of CCR1, CXCR3 and CX3CR1. All three purified monocyte subsets were infected by HTLV-1. The level of viral DNA in monocyte subsets correlated directly with their migration capacity to CCL2, CCL5 and CX3CL1 for the classical subset, with lesser phagocytosis for the intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+T-cells for the non-classical subset. These data suggest a model whereby HTLV-1 infection augments classical monocytes migration to tissues resulting in their infection, and non-classical monocytes frequency, resulting in increased transmission of virus to CD4+ and CD8+T-cells. Our data in humans together with prior animal experiments supports the notion that infection of monocytes in vitro is crucial for viral infectivity and persistence in humans, rendering infected monocytes desirable therapeutic targets.

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.

Incidence of tropical spastic parapesis/ HTLV-1 associated myelopathy/ (TSP/HAM) in patients followed in a Reference center in Salvador, Brasil

It is estimated that currently 5 to 10 million people are infected with HTLV-1 worldwide. In Brazil, Salvador is the city with the highest prevalence of HTLV-1 (2% in women and 1.2% in men). Tropical spastic paraparesis/ HTLV associated myelopathy (TSP/HAM) is an insidious and disabling chronic neurological disease. The incidence of TSP/HAM varies in different geographical regions with low rate in Japan and Caribbean islands and higher in Minas Gerais, Brazil. The incidence of TSP/HAM among patients attending the CHTLV in Salvador/BA, from 22 to 2013 was estimated. The outcome of interest was the evolution from asymptomatic status for TSP/HAM diagnosis, which was based on the Belem criteria. The follow-up for individuals that remained asymptomatic was the time between the year of the last medical consultation and the year of the HTLV-1 serological diagnosis and for those that developed TSP/HAM the time between the year of diagnosis of TSP/HAM and the year of HTLV-1 serological diagnosis. The exclusion criteria were a diagnosis of TSP/HAM at the first medical consultation, records with incomplete data and TSP/HAM possible diagnosis. From a sample of 243 patients, 91 were included. Two patients developed TSP/HAM definite and four had TSP/HAM probable diagnosis. The incidence density of TSP/HAM definite was 6.9 and 13 per 1,0 HTLV-1 infected individuals per year, respectively. The cumulative incidence to TSP/HAM definite was 2.29% while to TSP/HAM probable was 4.39%. The incidence rate found herein was higher than that observed in previous studies in Japan and Caribbean, however, similar to that reported in Brazil. More accurate cohort studies should be conducted to measure the actual risk of developing this disease.

Consequences of the association between HTLV-1 and tuberculosis

HTLV-1 modifies the immune response and clinical presentation of bacterial, fungal and helminthic infections. There are also evidences that helminthic infections may modify disease expression related to HTLV-1, increasing the occurrence of Adult T cell leukemia (ATL) and decreasing the development of HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). Recently it has been shown that HTLV-1 increases susceptibility for tuberculosis (TB) but the consequences of this co-infection in the clinical course of TB and HTLV-1 infection is not so clear. The aim of this study was to evaluate if TB influences neurologic disease in HTLV-1 infected subjects and to evaluate sequels of TB in HTLV-1 infected individuals. This is a nested case control study with the participation of 32 patients with previous diagnosis of TB and positivity sorology for HTLV-1 and 32 controls with HTLV-1 without TB paired by age, gender and time of admission in the clinic. Of the 32 patients with TB 29 had pulmonary TB, one had pleural TB, one laryngeal TB and the other lymphonode TB. Only 2 (6,2%) developed a second episode of TB. Among those who have been treated for TB 8 (25%) patients complained of dyspnea, 3 without evidence of pulmonary disease, 3 cases had chronic obstructive pulmonary disease (COPD) and two cases had bronchial overactivity. Regarding HTLV-1 associated disease probable HAM/TSP was diagnosed in 4 (12,5%) of the TB patients and in 6 (18,7%) of the patients who only have HTLV-1 infection (P > 0.05). Definitive HAM/TSP was diagnosed in 10 (31,2%) of the co-infected patients with HTLV-1 and TB and in 4 (12,5%) in the group who only had HTLV-1 (P < 0.05). While HTLV-1 appears to not influence the severity of Micobacterium tuberculosis infection TB was highly associated with HAM/TSP.

HTLV-1-induced leukotriene B4 secretion promotes the recruitment of target cells

Isolated HTLV-1 viral particles are poorly infectious both in vivo and in vitro. In contrast, the virus is efficiently transmitted through cell-to-cell contact. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied, the mechanisms facilitating the encounter between an infected cell and a target cell remain unknown. We postulated that the infected cells could behave as a source of chemoattractant, thus recruiting target cells. In 2012, Trindade et al. showed that PBMCs from HTLV-1 infected individuals (HTLV-1 asymptomatic carrier and HAM/TSP patients) could secrete higher levels of leukotriene B4 (LTB4), a potent chemoattractant, than cells from healthy donors. We proposed that, although T cells purportedly secrete no LTB4, LTB4 could be secreted by HTLV-1 infected lymphocytes and facilitate cell-to-cell contacts and viral transmission. We showed by ELISA that HTLV-1 infected lymphocytes (both primary CD4+ T cells and chronically infected cell lines) secrete LTB4. By cell transduction with lentiviral vectors encoding Tax, we suggest that HTLV-1 induced LTB4 secretion is a consequence of Tax-induced cytosolic phospholipase A2 (cPLA2) over expression. Blocking LTB4 secretion (with diverse specific inhibitors of the leukotriene pathway) prevented the recruitment of potential target cells, as well as the formation of cell-to-cell contacts. In conclusion, HTLV-1 diverts the leukotriene pathway and LTB4-mediated chemotaxis facilitates viral propagation.

Comparative analysis of gene expression patterns in the HTLV-1 infected T-cell clones

The integration of Human T-lymphotropic virus type I (HTLV-1) is known to be diverse but biased towards genomically active regions. Each infected clone is identified by a unique integration site position. The integration site position is known to be associated with the expression pattern of the provirus and its propensity to expand in vivo but it is not known how it would impact the gene expression of the host genes. The viral transactivator Tax has been shown (mostly in vitro) to be capable of altering the expression of many host genes however it is not known what role the genomic integration site may have in determining the alteration of host gene expression or how the gene expression pattern would vary from one infected T-cell clone to another. We have developed a panel of HTLV-1 infected T-cell clones. CD4+ T-cells were isolated from HTLV-1 infected individuals and cloned by limiting dilution. Each clone was cultured in the presence of integration inhibitor to prevent super infection and defined by a single unique integration site. Uninfected T cell clones were also established in the same way. In order to address this question and to analyze gene expression pattern, total RNAs were isolated from each clone for RNA sequencing (RNA-seq). We will present the results of high-throughput sequencing of the human and viral mRNA, highlighting the similar and variable between the different clones. # Poster award winnder - 2nd place

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.

Northern African strains of human T-lymphotropic virus type 1 arose from a recombination event

Although recombination is a major source of genetic variability in retroviruses, no recombinant strain had been observed for HTLV-1, the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype A is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, including the complete LTR and a 522-bp fragment of the env gene, which we collected from 19 countries throughout North, West and Central Africa, the continent where the virus has the largest endemic presence. The samples were obtained from 41 HTLV-1 infected individuals with different clinical statuses including ATL, TSP/HAM and asymptomatic carriers. The recombinant search and breakpoint detection were performed by boot scanning in Simplot to compare inferred clusters of sequences to each other. Finally, molecular clock analyses were performed to date the recombinant event observed. This led us to demonstrate the presence of recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,0 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus (Desrames et al., J. Virology, 88, 2014). In order to precise the geographical distribution of this recombinant within the African Continent, we studied a new set of samples from HTLV-1 infected patients of diverse African origin. The data on this on-going study will be presented.