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

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.

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 neurologic diseases in a patient with primate T lymphotropic virus type 1 (PTLV-1).

BACKGROUND: Virus transmission from various wild and domestic animals contributes to an increased risk of emerging infectious diseases in human populations. HTLV-1 is a human retrovirus associated with acute T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 originated from ancient zoonotic transmission from nonhuman primates, although cases of zoonotic infections continue to occur. Similar to HTLV-1, the simian counterpart, STLV-1, causes chronic infection and leukemia and lymphoma in naturally infected monkeys, and combined are called primate T-lymphotropic viruses (PTLV-1). However, other clinical syndromes typically seen in humans such as a chronic progressive myelopathy have not been observed in nonhuman primates. Little is known about the development of neurologic and inflammatory diseases in human populations infected with STLV-1-like viruses following nonhuman primate exposure. RESULTS: We performed detailed laboratory analyses on an HTLV-1 seropositive patient with typical HAM/TSP who was born in Liberia and now resides in the United States. Using a novel droplet digital PCR for the detection of the HTLV-1 tax gene, the proviral load in PBMC and cerebrospinal fluid cells was 12.98 and 51.68 %, respectively; however, we observed a distinct difference in fluorescence amplitude of the positive droplet population suggesting possible mutations in proviral DNA. A complete PTLV-1 proviral genome was amplified from the patient's PBMC DNA using an overlapping PCR strategy. Phylogenetic analysis of the envelope and LTR sequences showed the virus was highly related to PTLV-1 from sooty mangabey monkeys (smm) and humans exposed via nonhuman primates in West Africa. CONCLUSIONS: These results demonstrate the patient is infected with a simian variant of PTLV-1, suggesting for the first time that PTLV-1smm infection in humans may be associated with a chronic progressive neurologic disease.

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

UNLABELLED: 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.

OBJECTIVE: 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). METHODS: SCCSA and clinical disability scores were measured in 18 HAM/TSP patients, 4 asymptomatic carriers (ACs) of HTLV-1, 18 MS patients, and 10 HVs from a 3T magnetic resonance imaging. SCCSA measured in patients and ACs were compared to that of HVs and correlated with disability scores. RESULTS: The entire spinal cord in HAM/TSP patients was thin compared to HVs, whereas only the cervical cord in MS patients was thinner than in HVs (p < 0.0001). In HAM/TSP patients, SCCSA extensively correlated with Ambulation Index, whereas only the cervical cord correlated with disease duration (p < 0.05). In MS patients, the SCCSA extensively correlated with Scripps Neurologic Rating Score and the Expanded Disability Status Scale (p < 0.05). One of the 4 ACs showed atrophy in a pattern similar to HAM/TSP. INTERPRETATION: These results are in accordance with the findings that whereas over half of all lesions in an MS cord are seen in the upper cervical cord, most of the pathology in HAM/TSP is seen in the thoracolumbar cord, which in turn may be responsible for the more extensive cord atrophy seen in HAM/TSP. An imaging marker such as SCCSA might serve as a surrogate endpoint in clinical trials, especially to assess the neuroprotective impact of various therapies.

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.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Clinical trial of raltegravir, an integrase inhibitor, in HAM/TSP.

OBJECTIVE: Human T-cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive myelopathy. A high proviral load (PVL) is one of the main risk factors for HAM/TSP. Recently, it was shown that raltegravir could inhibit cell-free and cell-to-cell transmission of HTLV-1 in vitro. Given the substantial clinical experience in human immunodeficiency virus infection and its excellent safety profile, this agent may be an attractive therapeutic option for HAM/TSP patients. METHODS: Sixteen subjects with HAM/TSP received raltegravir 400 mg orally twice daily in an initial 6-month treatment phase, followed by a 9-month post-treatment phase. HTLV-1 PVLs were assessed using droplet digital PCR from the PBMCs every 3 months, and from the CSF at baseline, month 6, and month 15. We also evaluated the ability of raltegravir to regulate abnormal immune responses in HAM/TSP patients. RESULTS: While a downward trend was observed in PBMC and/or CSF PVLs of some patients, raltegravir overall did not have any impact on the PVL in this HAM/TSP patient cohort. Clinically, all patients' neurological scores and objective measurements remained relatively stable, with some expected variability. Immunologic studies showed alterations in the immune profiles of a subset of patients including decreased CD4+ CD25+ T cells and spontaneous lymphoproliferation. INTERPRETATION: Raltegravir was generally well tolerated in this HAM/TSP patient cohort. A subset of patients exhibited a mild decrease in PVL as well as variations in their immune profiles after taking raltegravir. These findings suggest that raltegravir may be a therapeutic option in select HAM/TSP patients. CLINICAL TRIAL REGISTRATION NUMBER: NCT01867320.

Clinical trial of a humanized anti-IL-2/IL-15 receptor ß chain in HAM/TSP.

OBJECTIVE: Human T cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neurological disease. Chronic activation of CD8+ T cells, as evidenced by increased spontaneous lymphoproliferation and HTLV-1-specific cytotoxic T cells, has been demonstrated in HAM/TSP patients. Since IL-2 and IL-15 stimulate memory CD8+ T cell activity, these cytokines have been implicated in the immunopathogenesis of HAM/TSP. In this phase I trial, we evaluated the safety, pharmacokinetics, and ability of Hu-Mikß1, a humanized monoclonal antibody directed toward the IL-2/IL-15 receptor ß-chain (IL-2/IL-15Rß: CD122), to saturate CD122 and regulate abnormal immune responses in patients with HAM/TSP by inhibition of IL-15 action. METHODS: Hu-Mikß1 was administered intravenously at doses of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg in a total of nine HAM/TSP patients. Five doses of Hu-Mikß1 were administered at 3-week intervals. The clinical response was evaluated using standardized scales. Viral and immunologic outcome measures were examined including HTLV-1 proviral load, T cell phenotypic analysis and spontaneous lymphoproliferation in HAM/TSP patients. RESULTS: There was no significant toxicity associated with Hu-Mikß1 administration in HAM/TSP patients. Saturation of CD122 by Hu-Mikß1 was achieved in five out of nine HAM/TSP patients. Administration of Hu-Mikß1 was associated with inhibition of aberrant CD8+ T cell function including spontaneous lymphoproliferation and degranulation and IFN-γ expression, especially in HAM/TSP patients that achieved CD122 saturation. INTERPRETATION: The treatment with Hu-Mikß1 had a number of immunological effects on HAM/TSP patients although no clinical efficacy was observed. We also did not see any dose-related toxicity.

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

BACKGROUND: 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. METHODS: Five patients and 5 matched healthy volunteers (HVs) underwent MRI of the cervical and thoracic spinal cord at 1.5 T. Quantification of the spinal cord volume was obtained from 3-dimensional MR images using a semiautomatic technique based on level sets. An unpaired t-test was used to assess statistical significance. RESULTS: Significant differences were found between mean spinal cord volume of HVs and HAM/TSP patients. The thoracic spinal cord volume was 14,050 ± 981 mm(3) for HVs and 8,774 ± 2,218 mm(3) for HAM/TSP patients (P = .0079), a reduction of 38%. The cervical spinal cord volume was 9,721 ± 797 mm(3) for HVs and 6,589 ± 897 mm(3) for HAM/TSP patients (P = .0079), a reduction of 32%. These results suggest that atrophy is evident throughout the spinal cord not routinely quantified. CONCLUSIONS: Semiautomatic spinal cord volume quantification is a sensitive technique for quantifying the extent of spinal cord involvement in HAM/TSP.

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.