Breanna Caruso

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.

Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay.

The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences between HHV-6A and HHV-6B has led to their recent reclassification as separate species. As it is now especially relevant to investigate each virus, our objectives were to first design a multiplex HHV-6A and HHV-6B ddPCR assay and then to investigate the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and patients with MS, a disease in which HHV-6 is thought to play a role. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus, which has important implications in clinical settings. Interestingly, upon comparing the saliva from MS patients and healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva; increased detection of HHV-6A in MS patients is consistent with other studies suggesting that this viral species (thought to be more neurotropic than HHV-6B) is more prevalent among MS patients compared to healthy donors. As the biology and disease associations between these two viral species differ, identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of each in health and disease.

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.

Dose-adjusted EPOCH chemotherapy with bortezomib and raltegravir for human T-cell leukemia virus-associated adult T-cell leukemia lymphoma

Dose-adjusted EPOCH chemotherapy with bortezomib and raltegravir for human T-cell leukemia virus-associated adult T-cell leukemia lymphoma

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.

Herpesvirus trigger accelerates neuroinflammation in a nonhuman primate model of multiple sclerosis

Significance Inflammatory processes drive the autoimmune disease multiple sclerosis (MS). However, what triggers this inflammation remains unknown. Several herpesviruses (HHVs), such as HHV-6 typically acquired during childhood, are associated with MS. The temporal separation between HHV-6 acquisition and MS development complicates its study as a disease trigger. Because rodents are not susceptible to HHV-6 infection, we utilized nonhuman primates to examine the impact of HHV-6 infection on an experimental MS-like disease. The viral infections were asymptomatic; however, the MS-like disease was significantly accelerated in all virally inoculated animals. Our data support the hypothesis that viruses may act as triggers to lower the threshold for autoimmunity, and warrant trials of antiviral interventions in early disease stages. Pathogens, particularly human herpesviruses (HHVs), are implicated as triggers of disease onset/progression in multiple sclerosis (MS) and other neuroinflammatory disorders. However, the time between viral acquisition in childhood and disease onset in adulthood complicates the study of this association. Using nonhuman primates, we demonstrate that intranasal inoculations with HHV-6A and HHV-6B accelerate an MS-like neuroinflammatory disease, experimental autoimmune encephalomyelitis (EAE). Although animals inoculated intranasally with HHV-6 (virus/EAE marmosets) were asymptomatic, they exhibited significantly accelerated clinical EAE compared with control animals. Expansion of a proinflammatory CD8 subset correlated with post-EAE survival in virus/EAE marmosets, suggesting that a peripheral (viral?) antigen-driven expansion may have occurred post-EAE induction. HHV-6 viral antigen in virus/EAE marmosets was markedly elevated and concentrated in brain lesions, similar to previously reported localizations of HHV-6 in MS brain lesions. Collectively, we demonstrate that asymptomatic intranasal viral acquisition accelerates subsequent neuroinflammation in a nonhuman primate model of MS.

Patterns of spinal cord atrophy in HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP)

Spinal cord inflammation and atrophy contribute to debilitating symptoms in HAM/TSP. We have developed a robust and fast algorithm to determine average cross-sectional area in cervical (c-spine) and thoracic (t-spine) spinal cords by tracing contours perpendicular to the edge in T1-weighted MRI images. The cross-sectional areas in the c- and t-spines were determined in 25 HAM/TSP, 10 asymptomatic carriers (AC) and 10 healthy volunteer (HV) subjects. To date, we have followed 8 of the HAM/TSP patients longitudinally over a two-year period. When compared to the HV data, the HAM/TSP spinal cord profiles fell into four general categories: atrophic entire spine (48%), atrophic t-spine (32%), atrophic c-spine (8%), and normal (12%). The majority of ACs had similar spinal cord profiles to those in the HV group, however, 3 ACs showed a pattern similar to HAM/TSP. As a group, both HAM/TSP c- and t-spines were significantly lower than those of HV (p<0.01). In the 8 patients with follow-up scans, spinal cord size showed an overall decreasing trend over time. In a rapidly progressing patient with the shortest disease duration, we could estimate spinal cord atrophy at a rate of 11% a year in the thoracic cord. In addition, change in proviral load negatively correlated with change in both c- and t-spine cross-sectional area (p<0.05) for patients with shorter disease duration and increasing proviral loads (i.e. an increase in proviral load was associated with a more atrophic cord). These results suggest that the pattern of spinal cord tissue damage is specific to the underlying inflammatory disease, a finding that has direct implications for the use of average cross-sectional spinal cord area as a surrogate end point for clinical trials.

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.

Detection of HTLV-1 replication and CD4+ proliferation in the humanized BLT mouse model

Human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic and the mechanisms driving disease remain poorly understood. Here we report our findings of HTLV-1 infection in humanized nonobese diabetic (NOD) severe combined immunodeficiency (SCID) common gamma chain knockout (NSG) mice that have been implanted with fetal bone marrow-liver-thymus tissue (BLT). BLT mice were injected with lethally irradiated CD4+ HTLV-1 producing cells. Blood samples were collected every four weeks to monitor CD4 and CD8 phenotypes and DNA isolation. Concomitant with an increase in the number of CD4+CD25+ T-lymphocytes in exposed animals compared to controls, we observed a significant increase in the HTLV-1 viral DNA load. Surprisingly, unlike what we observed in macaques, HTLV-1 virus mutated to knockout Orf-I protein expression was able to infect BLT mice and high viral DNA loads were detected. However, after sequencing the orf-I region of proviral DNA from infected animals, we found reversion of the point mutation to wild type. This was observed as early as four weeks after exposure, indicating in vivo viral replication and selection for Orf-I expression. Thus, the BLT mouse model successfully recapitulates HTLV-1 infection and may serve as an important tool for investigating in vivo mechanisms of HTLV-1 disease pathology and in evaluating drugs and treatment strategies.