Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity

The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vβ gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the Vβ gene usage were determined by comparing the TCR Vβ gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro . Diverse Vβ genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro . Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vβ gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo , the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development.     

Seroindeterminate HTLV-1 Prevalence and Characteristics in Blood Donors in Taiwan

Human T-cell leukemia virus type 1 (HTLV-1) is commonly accepted as the cause of adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. Screening of blood donors for HTLV-1 and HTLV-2 was implemented in Taiwan in February 1996. From February 1996 to December 1998, we investigated the seroprevalence of HTLV-1 in all unpaid blood donors in Taiwan. Of 2,578,238 donors in all 6 blood centers, 1793 (0.06%) were seropositive for HTLV-1, and 605 (0.023%) were indeterminate for HTLV-1. Among these indeterminate donors, 359 (59.3%) were male. The most common HTLV-1-indeterminate pattern by Western blot in our study was GD21 alone (34.6%) followed by p24 alone (7.8%), p53 alone (6.5%), and gp46 + GD21 (6.0%). That GD21 pattern was found in 59.6% of indeterminate results in this study suggested that the majority of nonspecific enzyme immunoassay reactions were probably precipitated by viral envelop glycoprotein GD21.     

Molecular aspects of HTLV-1 entry: functional domains of the HTLV-1 surface subunit (SU) and their relationships to the entry receptors

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage.     

Research and discovery of the first human cancer virus, HTLV-1

Human T-cell lymphoma virus (HTLV)-1 was the first human retrovirus to be discovered. It has been recognized as the cause of adult T-cell leukemia (ATL). In addition to giving a historical perspective on HTLV-1 and other retrovirus research, this paper discusses the origin of HTLV-1; the modes of transmission and global epidemiology of HTLV-1 infection; the genome of HTLV-1 and the mechanism of HTLV-1-induced leukemogenesis; the role of HTLV-1 in other diseases, and recent breakthroughs in ATL therapy.     

Pseudotypes of human T-cell leukemia virus types 1 and 2: neutralization by patients'' sera.

Pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of human T-cell leukemia virus (HTLV) types 1 and 2 were prepared by propagating VSV in cells lines productively infected with HTLV. Plaque assays of VSV (HTLV) pseudotypes were employed to determine the presence of (i) HTLV receptors on cells and (ii) neutralizing antibodies in the serum of patients with adult T-cell leukemia-lymphoma (ATLL). Cell surface receptors for HTLV-1 and HTLV-2 were found on nonlymphoid cells of human and mammalian origin. Neutralizing antibodies specific to VSV(HTLV-1) were found in sera of ATLL patients in titers varying from 1:50 to 1:30,000 and did not correlate closely with antibody titers for internal viral antigens. Sera from ATLL patients in the United Kingdom (Caribbean immigrants), United States, and Japan completely neutralized VSV (HTLV-1), indicating that the HTLV isolates from these distinct geographic regions represent a single envelope serotype. Neutralization of VSV (HTLV-1) was more specific and more sensitive than assays of syncytium inhibition. No cross-neutralization was observed between bovine leukosis virus and HTLV, and only limited cross-reaction was found for envelope antigens of HTLV-1 and HTLV-2. These studies show that VSV (HTLV) pseudotypes can be readily used to screen for neutralizing antibodies in patients' sera and to distinguish HTLV envelope serotypes.     

Current status of HTLV-1 infection

It is 30 years since human T-cell leukemia virus type 1 (HTLV-1) was identified as the first human retrovirus. To assess the implications of the virus for human health it is very important to know the past and present prevalence. Most of the estimates of HTLV-1 prevalence are based on serological screening of blood donors, pregnant women and other selected population groups. The widely cited estimate that the number of HTLV-1 carriers in Japan is 1.2 million was calculated from data that are now more than 25 years old. Here I summarize previous reports of prevalence studies in the world and Japan. Then, a recent analysis of seroprevalence of healthy blood donors in Japan will be described in comparison with that of 1988. A decrease in the number of HTLV-1 carriers in Japan was demonstrated, however, it is still more than one million. The number has increased in the metropolitan areas, probably reflecting the migration of Japanese population. I conclude that there is a paucity of general population data in countries where HTLV-1 is endemic, and re-evaluation of HTLV-1 infection is required to understand the virus burden on the human health.     

Monocytes from HTLV-1-infected patients are unable to fully mature into dendritic cells

Human T-cell lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1-associated myelopathy/tropical spastic paraparesis is a chronic inflammatory disease characterized by loss of motor movement in response to spinal marrow cell destruction by T lymphocytes. To perform their cellular function, T cells need to be activated by antigen-presenting cells, such as dendritic cells (DCs). The aim of this work was to analyze DC differentiation and activation from monocytes of HTLV-1-infected individuals. We demonstrated that monocytes from HTLV-1-infected patients who had been stimulated to differentiate had an impaired loss of CD14 expression, expressed low levels of CD1a, and maintained secretion of tumor necrosis factor-α compared with monocytes from noninfected donors. We further evaluated DC activation by tumor necrosis factor-α. We observed that in response to activation, DCs that were derived from noninfected donors had an increase in the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells, whereas in DCs derived from HTLV-1-infected patients, the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells remained similar to that of nonactivated cells. Moreover, these cells had an impaired capacity to stimulate allogeneic T lymphocytes. We demonstrated that DC maturation was altered in HTLV-1-infected patients, which could contribute to the development of HTLV-1-associated diseases.     

In vivo analysis of replication and immunogenicity of proviral clones of human T-lymphotropic virus type 1 with selective envelope surface-unit mutations

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo.     

Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy

Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We used a pool of 138 synthetic overlapping pentadecapeptides overspanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T cells that selectively recognized 1-3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide-loaded targets sharing single HLA class I or II alleles identified the restricting HLA alleles. HLA-A*0201+ donors consistently responded to pentadecapeptides containing HLA-A*0201-binding epitope(aa495-503)NLVPMVATV. T-cell lines from other donors contained high frequencies of CD4 and/or CD8 T cells selectively reactive against peptides presented by other HLA alleles, including both known epitopes such as (aa341-350)QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as (aa267-275)HERNGFTVL (HLA-B*4001 and B*4002) and (aa513-523)FFWDANDIYRI (HLA-DRB1*1301). These T cells consistently lysed CMV-infected target cells. Thus, this approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity that also lyse CMV-infected targets, and from a functional and regulatory perspective, may have advantages for generating virus-specific T cells for adoptive immunotherapy.     

Development of a Monoclonal Antibody Targeting HTLV-1 Envelope gp46 Glycoprotein and Its Application to Near-Infrared Photoimmuno-Antimicrobial Strategy

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.     

Factors predisposing to HTLV-1 infection in residents of the greater Tokyo area

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia. The geographic distribution of HTLV-1 carriers is quite uneven in Japan and the greatest prevalence is in southwestern Japan. Because many people move from endemic areas to the greater Tokyo area, the geographic distribution might have changed. Therefore, we investigated the factors predisposing to HTLV-1 infection, including birthplace, for 88 HTLV-1-infected individuals in greater Tokyo who visited our outpatient clinic. Of these, 39.5% were born in endemic areas, which include Kyushu/Okinawa, south Shikoku, Kii, Tohoku, and Hokkaido, whereas 38.3% were born in greater Tokyo and the proportion is presumed to be increasing. Half of the HTLV-1 infected individuals in greater Tokyo came from endemic areas, whereas around half of the remaining half was presumed to be involved in sexual transmission from a spouse from an endemic area. Overall, they constituted approximately 70% of the HTLV-1 carriers in greater Tokyo. These migration effects may increase the prevalence of HTLV-1 in the greater Tokyo area; nationwide surveillance is warranted.     

Immunizations of monkeys with synthetic peptides disclose conserved areas on gp120 of human immunodeficiency virus type 1 associated with cross-neutralizing antibodies and T-cell recognition.

Site-directed immunization was employed to identify sites on the envelope glycoprotein gp120 for antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-1). Antisera were raised in monkeys (Macaca fascicularis) against a series of 40 overlapping synthetic peptides covering the entire amino acid sequence of gp120 from the HTLV-IIIB strain of HIV-1. Immune sera against 12 of these peptides were reactive with gp120 by immunoblotting analysis, and antisera raised against 5 peptides, corresponding to amino acids (aa) 152-176, 193-218, 206-230, 248-269, and 307-330, were highly efficient in neutralizing HIV-1 (HTLV-IIIB) infectivity in vitro. Admixture of individual neutralizing anti-peptide monkey sera resulted in increment in neutralizing antibody titer. Antisera with reactivity to the relatively conserved regions defined by aa 152-176, 193-230, and 248-269 also neutralized to different extents the infectivity of the five Swedish clinical isolates of HIV-1 tested. Only a few HIV-1-infected people were found to make antibodies to these three conserved domains of gp120 as judged by ELISA using synthetic peptides as antigens. Three of the peptides (aa 152-176, 248-269, and 307-330) that induced neutralization antibodies also induced interleukin 2 production and lymphocyte proliferation when added to cultures of peripheral blood mononuclear cells from monkeys immunized with the corresponding peptides, indicating that these domains accommodate T-cell recognition sites. The results have obvious implications for the rational design of subunit vaccines against HIV-1 infection.     

Endocrine and metabolic disorders in HTLV-1 infected patients

Human T-cell leukemia virus type 1 (HTLV-1) infection is endemic in Japan and several countries in South America, Caribbean and Africa. Endocrine and metabolic disorders have been variably reported to be associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Therefore, the aim of this article was to critically evaluate the current knowledge of the endocrine and metabolic disorders associated with HTLV-1 infection. The literature search used PubMed, Web of Science, and LILACS databases in the past 10 years, utilizing, in various combinations, the following keywords: HTLV-1, adult T-cell leukemia, diabetes mellitus, GLUT-1, osteoporosis, hypercalcemia, autoimmune thyroid disorders, diabetes insipidus, inappropriate antidiuretic hormone secretion; pseudohypoparathyroidism; pseudopseudohypoparathyroidism. The proven endocrine manifestations of the HTLV-1 infection are calcium disorders which occur in some patients with acute HTLV-1/Adult T-cell leukemia/lymphoma. The few reports about thyroid, parathyroid, antidiuretic hormone and diabetes mellitus are insufficient to prove a causal association with HTLV-1 infection. The evidence for an association between endocrine disorders and HTLV-1 infection in general, and in asymptomatic patients is lacking. Given all these uncertainties, the endocrine expression of the HTLV-1 infection composes a promising research line for understanding the pathophysiology of this infection.     

Epitope mapping of human monoclonal antibodies recognizing conformational epitopes within HTLV type 1 gp46, employing HTLV type 1/2 envelope chimeras.

The majority of the antibody response to HTLV-1 surface glycoprotein, gp46, is directed at conformational epitopes. However, the regions of HTLV-1 gp46 that contain conformational epitopes are poorly defined. We previously reported on human monoclonal antibodies (hMAbs) to conformational epitopes within the HTLV-1 surface glycoprotein (gp46) that inhibit HTLV-1-mediated syncytium formation (Hadlock KG, Rowe J, Perkins S, et al.: J Virol 1997;71:5828-5840). To localize the conformational epitopes recognized by these antibodies, chimeric envelope proteins were constructed in which selected regions of the HTLV-1 envelope were replaced with the corresponding sequences from other members of the HTLV family of retroviruses. The chimeras were tested for reactivity with three hMAbs to conformational epitopes in HTLV-1 gp46, PRH-7A, PRH-3, and PRH-4, and one hMAb to a linear epitope, 0.5alpha. hMAb PRH-3 was specifically nonreactive with a chimera that replaced amino acids 32-36 of HTLV-1 gp46 and exhibited sharply reduced reactivity with a chimera that replaced amino acids 224-251 of HTLV-1 with the corresponding HTLV-2 sequence. hMAb PRH-4 was specifically nonreactive with a construct replacing amino acids 1-162 of HTLV-1 gp46 with the corresponding HTLV-2 sequence. Thus, HTLV-1 gp46 contains multiple conformational epitopes located in the amino-terminal portion of the protein.     

Bronchopneumonopathy in HTLV-1 associated myelopathy (HAM) and non-HAM HTLV-1 carriers]

Human T-cell leukemia virus type 1 (HTLV-1) causes not only adult T-cell leukemia (ATL), but also HTLV-1 associated myelopathy, a recently described slowly progressive spastic paraparesis, in its carrier state. Pulmonary involvement has been reported in HAM patients. Based on bronchoalveolar lavage or histological examination, the pulmonary involvement has been characterized by accumulation of T-cells, especially activated T-cells, in the lung. We reviewed our data on pulmonary involvement in HAM, which suggested that the characteristic pulmonary involvement observed in HAM was not restricted to HAM patients, but was also observed in non-HAM HTLV-1 carriers. Based on the data, we report that HAM is a systemic disease and that HTLV-1 causes characteristic pulmonary involvement, which we termed HTLV-1 associated bronchopneumonopathy (HAB).