Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

Human T-cell leukemia virus type 1 (HTLV-1) causes serious and intractable diseases in some carriers after infection. The elimination of infected cells is considered important to prevent this onset, but there are currently no means by which to accomplish this. We previously developed “virotherapy”, a therapeutic method that targets and kills HTLV-1-infected cells using a cytolytic recombinant vesicular stomatitis virus (rVSV). Infection with rVSV expressing an HTLV-1 primary receptor elicits therapeutic effects on HTLV-1-infected envelope protein (Env)-expressing cells in vitro and in vivo. Simian T-cell leukemia virus type 1 (STLV-1) is closely related genetically to HTLV-1, and STLV-1-infected Japanese macaques (JMs) are considered a useful HTLV-1 surrogate, non-human primate model in vivo. Here, we performed an in vitro drug evaluation of rVSVs against STLV-1 as a preclinical study. We generated novel rVSVs encoding the STLV-1 primary receptor, simian glucose transporter 1 (JM GLUT1), with or without an AcGFP reporter gene. Our data demonstrate that these rVSVs specifically and efficiently infected/eliminated the STLV-1 Env-expressing cells in vitro. These results indicate that rVSVs carrying the STLV-1 receptor could be an excellent candidate for unique anti-STLV-1 virotherapy; therefore, such antivirals can now be applied to STLV-1-infected JMs to determine their therapeutic usefulness in vivo.     

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.     

Molecular determinants of human T-lymphotropic virus type 1 transmission and spread

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.     

Molecular mechanisms of HTLV-1 infection and pathogenesis

Human T cell leukemia virus type 1 (HTLV-1) is an etiological pathogen of several human diseases, including adult T-cell leukemia (ATL), HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and inflammatory disorders such as uveitis and dermatitis. HTLV-1 spreads mainly through cell-to-cell transmission, induces clonal proliferation of infected T cells in vivo, and after a long latent period, a subset of HTLV-1 carriers develop ATL. Understanding the molecular mechanisms of infection and oncogenesis is important for the development of new strategies of prophylaxis and molecular-targeted therapies, since ATL has a poor prognosis, despite intensive chemotherapy. In this review, we will summarize recent progress in HTLV-1 research, and especially novel findings on viral transmission and leukemogenic mechanisms by two viral oncogenes, HBZ and tax.     

Monoclonally integrated HTLV type 1 in epithelial cancers from rabbits infected with an HTLV type 1 molecular clone

In addition to T cell leukemias and lymphomas, human T cell leukemia virus type 1 (HTLV-1) infection has been associated with nonhematologic malignancies and described as the cause of one case of small-cell lung carcinoma. Infected primary epithelial cells have been isolated from sweat gland and oral mucosae of HTLV-1-infected human patients. In the present study, epithelial neoplasms developed in two rabbits experimentally infected with a molecular clone of HTLV-1 (strain K30p). Serologic detection of anti-HTLV-1 and isolation of virus from blood lymphocytes at multiple time points postinjection established a course of chronic asymptomatic infection in both. One rabbit, infected for 5.5 years after intramuscular injection of HTLV-1 DNA, developed a thymoma having features of medullary differentiation. HTLV-1 provirus was detected in both thymocytes and neoplastic epithelium isolated discretely from the thymoma by laser capture microdissection. These findings provide the first experimental evidence of HTLV-1 disease after infection by HTLV-1 DNA injection. Endometrial adenocarcinoma occurred in a second rabbit 2.5 years after its inoculation with cell-associated virus. In this second case, an epithelial cell line derived ex vivo from a metastatic lesion produced virus in culture. In tumors from each of the two rabbits, the neoplastic epithelium was infected and harbored monoclonally integrated HTLV-1 provirus. Although monoclonal provirus integration alone does not establish retroviral cause of carcinogenesis unequivocally, these and other accumulating data indicate that there may be a role for HTLV-1 in diseases associated with infection of epithelia, including some epithelial cancers.     

Dermatologic lesions in asymptomatic blood donors seropositive for human T cell lymphotropic virus type-1

Dermatologic manifestations are quite common in patients with adult T cell leukemia/lymphoma and myelopathy/tropical spastic paraparesis associated with infection with human T cell lymphotropic virus type-1 (HTLV-1). In this study, we evaluated the dermatologic lesions of eligible blood donors in the state of Minas Gerais in Brazil who were seropositive but asymptomatic for infection with HTLV-1. The study population was composed of 128 HTLV-1-seropositive individuals and 108 seronegative controls. All individuals underwent a dermatologic evaluation. Biopsy specimens were obtained from abnormal and normal skin samples of seropositive individuals in an attempt to detect HTLV-1 in tissue samples by a polymerase chain reaction. Dermatologic alterations were observed more frequently in the seropositive group (adjusted odds ratio [OR] = 8.77, 95% confidence interval [CI] = 4.11-18.71). The most common skin diseases were dermatophytoses (adjusted OR = 3.32, 95% CI = 1.50-7.35), seborrheic dermatitis (OR = 3.53, 95% CI = 0.67-24.66), and acquired ichthyosis (P = 0.001). Virus was detected more frequently in abnormal skin samples. Dermatologic lesions probably related to HTLV-1 infection were diagnosed in eligible blood donors who were infected with this virus, who were previously considered to be asymptomatic carriers of HTLV-1.     

Human T-Cell Leukemia Virus Type 1 Envelope Protein: Post-Entry Roles in Viral Pathogenesis

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4⁺ T-cell malignancy, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neurological disease. Disease progression in infected individuals is the result of HTLV-1-driven clonal expansion of CD4⁺ T-cells and is generally associated with the activities of the viral oncoproteins Tax and Hbz. A closely related virus, HTLV-2, exhibits similar genomic features and the capacity to transform T-cells, but is non-pathogenic. In vitro, HTLV-1 primarily immortalizes or transforms CD4⁺ T-cells, while HTLV-2 displays a transformation tropism for CD8⁺ T-cells. This distinct tropism is recapitulated in infected people. Through comparative studies, the genetic determinant for this divergent tropism of HTLV-1/2 has been mapped to the viral envelope (Env). In this review, we explore the emerging roles for Env beyond initial viral entry and examine current perspectives on its contributions to HTLV-1-mediated disease development.     

HTLV-1, Immune Response and Autoimmunity

Human T-lymphotropic virus type-1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATL). Tropical spastic paraparesis/HTLV-1-associated myelopathy (PET/HAM) is involved in the development of autoimmune diseases including Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and Sjögren’s Syndrome (SS). The development of HTLV-1-driven autoimmunity is hypothesized to rely on molecular mimicry, because virus-like particles can trigger an inflammatory response. However, HTLV-1 modifies the behavior of CD4⁺ T cells on infection and alters their cytokine production. A previous study showed that in patients infected with HTLV-1, the activity of regulatory CD4⁺ T cells and their consequent expression of inflammatory and anti-inflammatory cytokines are altered. In this review, we discuss the mechanisms underlying changes in cytokine release leading to the loss of tolerance and development of autoimmunity.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

Is it necessary to test patients with immune thrombocytopenic purpura (ITP) for seropositivity to HTLV-1?

HTLV-111 (HIV-1) has been shown to be associated with thrombocytopenia of a type resembling immune thrombocytopenic purpura (ITP). HTLV-1 is a retrovirus similar to HIV-I (HTLV-III) in a number of features, such as CD4 tropism. It is responsible for several clinical entities, including adult T-cell leukemia/lymphoma. The relationship, if any, of HTLV-1 and thrombocytopenia has not been systematically studied. To determine how frequently ITP patients are commonly infected with HTLV-1, the following study was performed. Frozen serum samples from 123 randomly selected patients with ITP were thawed and tested for antibodies to HTLV-1 by enzyme-linked immunoabsorbent assay. Positives were confirmed by Western blot. Three patients were initially found to be positive for HTLV-1. One was a female of Caribbean ancestry, one was a male HIV-1+ patient, and one was an adolescent female with no known risk factors for HIV-1. The two females later tested negative for HTLV-1. As a screening program for HTLV-1 antibodies was not introduced into blood banks until November 1988, there may have been passive transfer of the virus from intravenous immunoglobulin that these patients had received. This study of a large number of ITP patients shows that it is extremely unlikely that they are infected with HTLV-1, and, therefore, it is unnecessary to screen ITP patients for seropositivity to HTLV-1.     

Anti-Apoptotic Effect of Tax: An NF-κB Path or a CREB Way?

The NF-κB pathway is intimately linked to the survival of mammalian cells, and its activation by Tax has consequently been considered important for human T-cell leukemia/lymphoma virus type 1 (HTLV-1)-infected cell resistance to death. Very little emphasis has been given to other mechanisms, although Tax regulates the expression and activity of several cellular genes. The finding that CREB protein is activated in HTLV-1 infected cells underlines the possibility that other mechanisms of survival may be implicated in HTLV-1 infection. Indeed, CREB activation or overexpression plays a role in normal hematopoiesis, as well as in leukemia development, and CREB is considered as a survival factor in various cell systems. A better understanding of the different molecular mechanisms used by Tax to counteract cell death will also help in the development of new therapeutic strategies for HTLV-1 associated diseases.     

Does HTLV-1 Infection Show Phenotypes Found in Sjögren’s Syndrome?

Viruses are a possible cause for Sjögren’s syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.     

Mother-to-Child Transmission of HTLV-1 Epidemiological Aspects,Mechanisms and Determinants of Mother-to-Child Transmission

Human T-cell Lymphotropic Virus type 1 (HTLV-1) is a human retrovirus that infects at least 5–10 million people worldwide, and is the etiological agent of a lymphoproliferative malignancy; Adult T-cell Leukemia/Lymphoma (ATLL); and a chronic neuromyelopathy, HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), as well as other inflammatory diseases such as infective dermatitis and uveitis. Besides sexual intercourse and intravenous transmission, HTLV-1 can also be transmitted from infected mother to child during prolonged breastfeeding. Some characteristics that are linked to mother-to-child transmission (MTCT) of HTLV-1, such as the role of proviral load, antibody titer of the infected mother, and duration of breastfeeding, have been elucidated; however, most of the mechanisms underlying HTLV-1 transmission during breast feeding remain largely unknown, such as the sites of infection and cellular targets as well as the role of milk factors. The present review focuses on the latest findings and current opinions and perspectives on MTCT of HTLV-1.     

Molecular mechanisms affecting HTLV type 1-dependent fusion at the cell membrane: implications for inhibiting viral transmission

Infection with human T cell leukemia virus type 1 is detected by screening programs and contact follow-up procedures. Where chronic infection results in overt pathology, this is treated largely symptomatically and control of transmission relies on physical and educational constraints. The poor infectious transmission rate of HTLV-1 has long been described but to date has not been exploited in preventative measures to combat the spread of the virus. We undertook to investigate some of the molecular steps involved in HTLV-1 cell-cell fusion, the main mechanism of transmission. We showed that poor transmission may relate in part to an inefficiency in adopting and maintaining a fusion competent conformation of the HTLV-1 envelope TM protein. In cell-cell fusion, this deficiency can be complemented by accessory molecules on both infected and target cells that stabilize the envelope/receptor interaction. In virion-cell fusion, this is less likely, leading to an inefficient interaction and poor infectious transmission by cell-free virus. A discussion of the accessory molecules involved in HTLV-1 fusion is presented. This weak envelope-dependent interaction with target cells in the host can be potently disrupted by peptides that destabilize the TM protein structure and significantly inhibit HTLV-1 fusion. These observations may be useful in the design of therapeutic agents to prevent HTLV-1 transmission.     

Reduced Foxp3 protein expression is associated with inflammatory disease during human t lymphotropic virus type 1 Infection

The Foxp3 protein is a specific marker of CD4(+)CD25(+) regulatory T (T(reg)) cells, and its expression is critical to their development and function. Several studies have demonstrated the dysregulation of Foxp3 expression during human inflammatory diseases. Infection with human T lymphotropic virus type 1 (HTLV-1) is associated with the development of a number of inflammatory conditions, including myelopathy, although the majority of individuals who are infected with HTLV-1 remain asymptomatic. To examine the role played by T(reg) cells in the development of inflammatory disease during HTLV-1 infection, we examined Foxp3 expression by flow cytometry. Our analysis showed that HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with a lower expression (compared with that in asymptomatic HTLV-1 carriers and healthy donors) of Foxp3 in peripheral-blood leukocytes. In individuals infected with HTLV-1, Foxp3 expression was inversely correlated with HTLV-1 tax proviral DNA load. These results suggest that impaired Foxp3 expression may contribute to the development of inflammatory disease during HTLV-1 infection.     

HTLV-1 infection in Papua New Guinea: evidence for serologic false positivity

Serum samples from 557 individuals participating in studies from four separate lowland and highland populations in Papua New Guinea exhibited consistently false-positive results for human T lymphotropic virus (HTLV) type 1 (10%) and human immunodeficiency virus (HIV) type 1 (5%) antibody in direct antiglobulin and agglutination assays. All serum samples were negative in competitive ELISAs and radioimmunoassays for HTLV-1 and HIV-1; selected samples of reactive sera were negative in an HTLV-2 competitive ELISA. Immunofluorescent antibody tests using HTLV-1 infected cells correlated poorly with ELISA results. None of the sera from Papua New Guinea neutralized vesicular stomatitis virus pseudotypes of HTLV-1. By Western blot analysis, only three serum samples were weakly reactive to HTLV-1 gag proteins. These studies suggest there is as yet no firm evidence of HTLV-1, HTLV-2, or HIV-1 infection in Papua New Guinea, although there may be a low prevalence.     

Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission

The human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-1-associated myelopathy. HTLV-1 is transmitted to T cells through the virological synapse and by extracellular viral assemblies. Here, we uncovered an additional mechanism of virus transmission that is regulated by the HTLV-1-encoded p8 protein. We found that the p8 protein, known to anergize T cells, is also able to increase T-cell contact through lymphocyte function-associated antigen-1 clustering. In addition, p8 augments the number and length of cellular conduits among T cells and is transferred to neighboring T cells through these conduits. p8, by establishing a T-cell network, enhances the envelope-dependent transmission of HTLV-1. Thus, the ability of p8 to simultaneously anergize and cluster T cells, together with its induction of cellular conduits, secures virus propagation while avoiding the host's immune surveillance. This work identifies p8 as a viral target for the development of therapeutic strategies that may limit the expansion of infected cells in HTLV-1 carriers and decrease HTLV-1-associated morbidity.