Raya Massoud

Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations

An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL—real-time PCR—has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe.

Humoral immune response to HTLV-1 basic leucine zipper factor (HBZ) in HTLV-1-infected individuals

Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. HTLV-1 basic leucine zipper factor (HBZ) gene has a critical role in HTLV-1 infectivity and the development of ATL and HAM/TSP. However, little is known about the immune response against HBZ in HTLV-1-infected individuals. In this study, we examined antibody responses against HBZ in serum/plasma samples from 436 subjects including HTLV-1 seronegative donors, asymptomatic carriers (AC), ATL, and HAM/TSP patients using the luciferase immunoprecipitation system. Immunoreactivity against HBZ was detected in subsets of all HTLV-1-infected individuals but the test did not discriminate between AC, ATL and HAM/TSP. However, the frequency of detection of HBZ-specific antibodies in the serum of ATL patients with the chronic subtype was higher than in ATL patients with the lymphomatous subtype. Antibody responses against HBZ were also detected in cerebrospinal fluid of HAM/TSP patients with anti-HBZ in serum. Antibody responses against HBZ did not correlate with proviral load and HBZ mRNA expression in HAM/TSP patients, but the presence of an HBZ-specific response was associated with reduced CD4+ T cell activation in HAM/TSP patients. Moreover, HBZ-specific antibody inhibited lymphoproliferation in the PBMC of HAM/TSP patients. This is the first report demonstrating humoral immune response against HBZ associated with HTLV-I infection. Thus, a humoral immune response against HBZ might play a role in HTLV-1 infection.

Human T Cell Leukemia Virus Type 1 Infection of the Three Monocyte Subsets Contributes to Viral Burden in Humans

ABSTRACT Because the viral DNA burden correlates with disease development, we investigated the contribution of monocyte subsets (classical, intermediate, and nonclassical monocytes) to the total viral burden in 22 human T cell leukemia virus type 1 (HTLV-1)-infected individuals by assessing their infectivity status, frequency, as well as chemotactic and phagocytic functions. All three monocyte subsets sorted from HTLV-1-infected individuals were positive for viral DNA, and the frequency of classical monocytes was lower in the blood of HTLV-1-infected individuals than in that of uninfected individuals, while the expression levels of the chemokine receptors CCR5, CXCR3, and CX3CR1 in classical monocytes were higher in HTLV-1-infected individuals than uninfected individuals; the percentage of intermediate monocytes and their levels of chemokine receptor expression did not differ between HTLV-1-infected and uninfected individuals. However, the capacity of intermediate monocytes to migrate to CCL5, the ligand for CCR5, was higher, and a higher proportion of nonclassical monocytes expressed CCR1, CXCR3, and CX3CR1. The level of viral DNA in the monocyte subsets correlated with the capacity to migrate to CCL2, CCL5, and CX3CL1 for classical monocytes, with lower levels of phagocytosis for intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+ T cells for nonclassical monocytes. These data suggest a model whereby HTLV-1 infection augments the number of classical monocytes that migrate to tissues and become infected and the number of infected nonclassical monocytes that transmit virus to CD4+ and CD8+ T cells. These results, together with prior findings in a macaque model of HTLV-1 infection, support the notion that infection of monocytes by HTLV-1 is likely a requisite for viral persistence in humans. IMPORTANCE Monocytes have been implicated in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected HTLV-1 DNA in all three monocyte subsets and found that infection impacts surface receptor expression, migratory function, and subset frequency. The frequency of nonclassical patrolling monocytes is increased in HTLV-1-infected individuals, and they have increased expression of CCR1, CXCR3, and CX3CR1. The viral DNA level in nonclassical monocytes correlated with the viral DNA level in CD4+ and CD8+ T cells. Altogether, these data suggest an increased recruitment of classical monocytes to inflammation sites that may result in virus acquisition and, in turn, facilitate virus dissemination and viral persistence. Our findings thus provide new insight into the importance of monocyte infection in viral spread and suggest targeting of monocytes for therapeutic intervention.

In vivo imaging of spinal cord atrophy in neuroinflammatory diseases

Spinal cord atrophy is prominent in chronic progressive neurologic diseases such as human T‐cell lymphotropic virus type 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) and multiple sclerosis (MS). Here we compared the spinal cord cross‐sectional area (SCCSA) in HAM/TSP and MS patients to that of healthy volunteers (HVs).

Epigenetic Modification of the FoxP3 TSDR in HAM/TSP Decreases the Functional Suppression of Tregs

HTLV-1 is a human retrovirus that is associated with the neuroinflammatory disorder HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). In these patients, HTLV-1 is primarily found in the CD4+CD25+ T cell subset (Regulatory T cells:Tregs), which is responsible for peripheral immune tolerance and is known to be dysfunctional in HAM/TSP. Recent evidence suggests that FoxP3 expression and function is determined epigenetically through DNA demethylation in the Treg-specific demethylated region (TSDR). We analyzed the methylation of the TSDR in PBMCs, CD4+ T cells, and CD4+CD25+ T cells from normal healthy donors (NDs) and HAM/TSP patients. We demonstrated that there is decreased demethylation in analyzed PBMCs and CD4+CD25+ T cells from HAM/TSP patients as compared to NDs. Furthermore, decreased TSDR demethylation was associated with decreased functional suppression by Tregs. Additionally, increased HTLV-1 Tax expression in HAM/TSP PBMC culture correlated with a concomitant decline in FoxP3 TSDR demethylation. Overall, we suggest that HTLV-1 infection decreases Treg functional suppressive capacity in HAM/TSP through modification of FoxP3 TSDR demethylation and that dysregulated Treg function may contribute to HAM/TSP disease pathogenesis.

HTLV-I-associated myelopathy/tropical spastic paraparesis: semiautomatic quantification of spinal cord atrophy from 3-dimensional MR images.

Human T‐cell lymphotropic virus type I (HTLV‐I)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a disabling neurological disorder characterized by inflammatory changes in the spinal cord. We used a semiautomatic technique to quantify spinal cord volume from 3‐dimensional MR images of patients with HAM/TSP.

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical spastic paraparesis

Significance IL-2 and IL-15, members of the gamma chain family of cytokines, are prominently deregulated in human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and underlie many of the characteristic immune abnormalities such as spontaneous lymphocyte proliferation, increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific CD8+ T lymphocytes (CTLs). In this in vitro study, we demonstrate that selective and simultaneous blockade of IL-2 and IL-15, with a γ-chain antagonistic peptide, reduces spontaneous lymphocyte proliferation (SP), STAT5 phosphorylation, and more important, the degranulation of CD8+ T cells and the frequency of HTLV-1-specific CTLs. Thus, selective cytokine blockade with antagonistic peptides might be a therapeutic approach in HAM/TSP and is potentially applicable to multiple other conditions in which cytokines are pathogenic. Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive inflammatory myelopathy occurring in a subset of HTLV-1-infected individuals. Despite advances in understanding its immunopathogenesis, an effective treatment remains to be found. IL-2 and IL-15, members of the gamma chain (γc) family of cytokines, are prominently deregulated in HAM/TSP and underlie many of the characteristic immune abnormalities, such as spontaneous lymphocyte proliferation (SP), increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific cytotoxic CD8+ T lymphocytes (CTLs). In this study, we describe a novel immunomodulatory strategy consisting of selective blockade of certain γc family cytokines, including IL-2 and IL-15, with a γc antagonistic peptide. In vitro, a PEGylated form of the peptide, named BNZ132-1-40, reduced multiple immune activation markers such as SP, STAT5 phosphorylation, spontaneous degranulation of CD8+ T cells, and the frequency of transactivator protein (Tax)-specific CD8+ CTLs, thought to be major players in the immunopathogenesis of the disease. This strategy is thus a promising therapeutic approach to HAM/TSP with the potential of being more effective than single monoclonal antibodies targeting either IL-2 or IL-15 receptors and safer than inhibitors of downstream signaling molecules such as JAK1 inhibitors. Finally, selective cytokine blockade with antagonistic peptides might be applicable to multiple other conditions in which cytokines are pathogenic.

Targeting the binding interface on a shared receptor subunit of a cytokine family enables the inhibition of multiple member-cytokines with selectable target spectrum.

Background: The current therapeutic strategy cannot suppress multiple cytokines in human disease. Results: We developed a new strategy to selectively block γc-family cytokines. Conclusion: Our novel strategy was proven effective both in vitro and in vivo. Significance: This approach will fill in a gap in the existing therapeutic strategy. The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.

Development of HTLV-1 associated myelopathy/tropical spastic paraparesis in a patient with simian T-lymphotropic virus type 1-like infection.

Virus transmission from various wild and domestic animals contributes to increased risk of emerging infectious diseases in human populations. HTLV-1 is a human retrovirus associated with acute T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which originated from zoonotic transmission from various African and Asian nonhuman primates (NHPs). Similar to HTLV-1, the simian counterpart, STLV-1, causes chronic infection and leukemia and lymphoma in naturally infected monkeys. However, other clinical syndromes typically seen in human such as a chronic progressive myelopathy have not been observed in NHPs. Little is also known about the development of any neurologic and inflammatory diseases in human populations infected with STLV-1-like viruses following NHP exposure. We identified and analyzed the complete genome of a primate T lymphotropic virus type 1 (PTLV-1) isolated from a patient with typical HAM/TSP who resides in the United States but was born in Liberia. Using a novel droplet digital PCR for the detection of the HTLV-1 tax gene, the proviral load in PBMC was 14.01%; however there was a distinct difference in fluorescence amplitude compared to all other H!M/TSP patients, suggesting viral heterogeneity. A complete PTLV-1 proviral genome was amplified from DNA extracted from the PBMCs of the HAM/TSP patient using PCR to generate nine overlapping subgenomic fragments. Phylogenetic analysis of PTLV-1 env and LTR regions showed the virus was highly related with PTLV-1 from sooty mangabey monkeys and humans exposed from NHPs in West Africa. These results suggest the patient is likely infected with STLV-1, suggesting for the first time that viral transmission from monkey to human may be associated with a chronic progressive neurologic disease.

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.