Estimation of the window period of human T-cell leukemia virus type 1 and 2 tests by a lookback study of seroconverters among Japanese voluntary blood donors.

BACKGROUND: Japan is endemic for human T-cell leukemia virus type 1 (HTLV-1), and the horizontal transmission of HTLV-1 is often reported. However, the window period (WP) for serologic or molecular screening is unclear. STUDY DESIGN AND METHODS: Results for anti-HTLV-1 screening and confirmatory tests obtained from 648 591 repeated blood donors in the Kyushu district, one of the most endemic areas of HTLV-1 in the world, were evaluated. A lookback study was conducted for seroconverters. RESULTS: During 2012 to 2019, 436 seroconverters (155 men, 281women) were identified with use of a screening chemiluminescence enzyme-immunoassay (CLEIA) and multiple confirmatory tests. Because the period between the latest seronegative donation and seroconversion was highly variable (2.1-276.7 months), 19 cases that seroconverted within 6 months were subjected to the analysis. The WP of the particle agglutination assay and CLEIA was estimated to be 2.2 ± 0.6 and 2.6 ± 1.7 months, respectively. The WP of the indirect immunofluorescence assay was 4.8 ± 6.5 months. Although the WP of western blotting was estimated to be 6.3 ± 8.7 months, four cases were still indeterminate through the study period. Chemiluminescence and line immunoassays, the current screening and confirmatory tests used in the Japanese blood program, showed the shortest WP of 2.2 ± 0.6 months. The WP of real-time polymerase chain reaction for HTLV-1 was estimated to be 4.1 ± 7.8 months. CONCLUSIONS: The WP in commercially available testing systems for HTLV-1/2 was determined for natural infection among repeated blood donors. Considering the HTLV-1 WP will help increase transfusion safety and facilitate the accurate diagnosis of HTLV-1 infection.

Loss of the ex vivo but not the reinducible CD8+ T-cell response to Tax in human T-cell leukemia virus type 1-infected patients with adult T-cell leukemia/lymphoma.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). In asymptomatic carriers and HAM patients, HTLV-1 infection leads to a vigorous cytotoxic T-cell (CTL) response mainly directed to the regulatory Tax protein. In contrast, initial studies showed that anti-HTLV-1 CTL activities were not reproductively detected in ATLL patients, neither ex vivo, nor after in vitro restimulation. To better understand this discrepancy, we explored the anti-HTLV-1 CD8+ T-cell response of eight ATLL patients by using in vitro restimulated or freshly isolated CD8+ T cells. In all the ATLL patients, we found that mitogenic activation allowed the induction of CD8+ T cells able to lyse autologous HTLV-1-infected cells and/or to produce IFNgamma in response to Tax peptides. In contrast, only a minority of the patients possessed CD8+ cells able to respond ex vivo to the same epitopes. These findings indicate that although a restimulatable anti-HTLV-1 CTL activity persists during ATLL, the specific ex vivo response is not constantly maintained. This provides definitive evidence that the CD8+ T-cell response to HTLV-1 is affected by ATLL development and reveals that a major defect concerns the generation and/or the functionality of CD8+ effectors.

Pathological and virological assessment of acute HTLV-I-associated myelopathy complicated with encephalopathy and systemic inflammation.

HTLV-I-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP), is a chronic inflammatory disease of the spinal cord. Acute cases are uncommon. We report the case of a 41-year-old woman with acute HAM/TSP complicated with encephalitis, an intense inflammatory reaction of the nervous system and lymphocytic infiltration of skeletal muscles, liver, salivary, adrenal and pituitary glands. The immunohistochemical studies of the lymphocytes surrounding blood vessels showed both B- and T-lymphocytes, in similar proportion, with both CD4- and CD8-positive cells. In addition, many perivascular and scattered macrophages were observed. Adult T-cell leukemia/lymphoma (ATL) was ruled out. The marrow aspirate was normal. Serial cerebrospinal fluid (CSF) analysis showed presence of HTLV-I antibodies, but without intrathecal synthesis of specific antibodies. Determination of HTLV-I viral loads demonstrated increased levels in the CSF relative to the peripheral blood and may be associated with widespread inflammation. The pathological and immunological findings may help understand the role of immune-reactive cells in the pathogenesis of HTLV-I-associated myelopathy.

Human T cell leukemia virus Type I (HTLV-I) infection induces greater expansions of CD8 T lymphocytes in persons with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers.

A quantitative study of the T cell receptor repertoire was performed ex vivo on CD4 and CD8 T cell subsets of human T cell leukemia virus type I (HTLV-I)-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Indexes of oligoclonality that compiled all repertoire modifications were calculated for peripheral blood mononuclear cells and for CD4 and CD8 T cell subsets. Both patients with HAM/TSP and asymptomatic carriers had greater T lymphocyte expansions than did uninfected donors, which was independent of age and at least twice higher in the CD8 than in the CD4 cell compartment. Some expanded CD8 T cells corresponded to cytotoxic T lymphocytes directed against various epitopes of the immunodominant Tax protein. Patients with HAM/TSP had significantly higher CD8 cell expansions than did asymptomatic carriers. These results highlight the prognostic value of measuring CD8 T cell expansions during follow-up of HTLV-I infection.

Transmission of human T-cell leukaemia virus type I into adult mice.

Human T-cell leukaemia virus type I (HTLV-I)-transformed rabbit T-cells, F647a, were intraperitoneally injected into eight 10-week-old C3H/He and C3H/HeJ mice (1 x 10(7) F647a cells/mouse), respectively. Antibody titres against HTLV-I increased to a peak at 1-3 months after injection in both C3H/He and C3H/HeJ mice. At 12 months after injection, antibody titres of two of the eight C3H/HeJ mice became undetectable, whereas those of all the C3H/He mice still ranged from 1:10 to 1:40. Sera from both seropositive C3H/He and C3H/HeJ mice reacted with HTLV-I core proteins, but not with the env protein. HTLV-I proviral sequences were detected in two of eight C3H/He mice and three of the eight C3H/HeJ mice. These results suggest that HTLV-I is able to infect an adult mouse.

Antigenic variability of an immunodominant region (239-261) of the surface glycoprotein of HTLV-I.

In the 239-261 region of the surface glycoprotein of HTLV-I, we delineated five epitopes recognized by antibodies present in sera from HTLV-I infected patients. Three epitopes are located between the amino acids 252 and 261, one is of a linear type and the two others of a conformational type. One epitope is comprised between amino acids 244 and 249. The last epitope described lies between the amino acids 244 and 257 and strictly requires the presence of a serine at position 250 for its recognition. When patients are infected by viral isolates presenting a substitution of the serine at position 250 by a proline, no antibodies recognizing the 244-257 region could be found. Altogether, our data demonstrate that the immunodominant 239-261 region is complex and is subject to antigenic variability.

Long-term persistence of protective immunity in cynomolgus monkeys immunized with a recombinant vaccinia virus expressing the human T cell leukaemia virus type I envelope gene.

To develop effective vaccines against infection with human T cell leukaemia virus type I (HTLV-I), we constructed a recombinant vaccinia virus (WR-SFB5env) synthesizing the HTLV-I envelope (Env) gp46 protein under the control of a strong promoter, termed the ATI hybrid promoter. WR-SFB5env expressed a large quantity of gp46. In cynomolgus monkeys (Macaca fascicularis) immunized with WR-SFB5env, anti-HTLV-I Env antibody, including neutralizing antibody, was induced and remained at a high level until 136 weeks (2-6 years) post-infection (p.i.). These immunized monkeys had HTLV-I Env-specific cytotoxic T lymphocyte activity. At 136 weeks p.i., the immunized monkeys were challenged with an HTLV-I-producing cynomolgus T lymphocyte cell line. Neither HTLV-I antigen nor HTLV-I proviruses were detected in peripheral blood mononuclear cells, lymph nodes or spleens of the WR-SFB5env-immunized monkeys, in contrast to non-immunized control monkeys. These results indicate that a single immunization with WR-SFB5env induced prolonged humoral and cellular immune responses to HTLV-I and protected the monkeys against virus challenge.

The value of csf analysis for the differential diagnosis of HTLV-I associated myelopathy and multiple sclerosis

Amostras do líquido cefalorraquidiano (LCR) e soro de 17 pacientes brasileiros com HAM/TSP, seis com esclerose múltipla e seis com epilepsia idiopática (controle nao-inflamatório) foram analisadas para a presença de anticorpos para o vírus de sarampo, rubéola, varicela zoster e herpes simples pelo método de ELISA. Todos os casos de HAM/TSP e esclerose múltipla tinham resposta imune poliespecífica intratecal para sarampo e rubéola. Anticorpos específicos para sarampo e rubéola (resposta MRZ) foram observados em todos os pacientes com esclerose múltipla, mas nao nos controles com epilepsia idiopática. A relevância das respostas poliespecífica e monoespecífica é discutida para essas doenças neurológicas crônicas.

A soluble form of tumour necrosis factor receptor in cerebrospinal fluid and serum of HTLV-I-associated myelopathy and other neurological diseases.

Paired samples of cerebrospinal fluid (CSF) and serum from 17 patients with human T-cell lymphotrophic virus I (HTLV-I)-associated myelopathy, 5 patients with multiple sclerosis and 11 controls with non-inflammatory disorders (migraine, idiopathic epilepsy and myelopathy of unknown aetiology) were examined by enzyme-linked immunosorbent assay for the presence of the 60-kDa soluble form of tumour necrosis factor receptor (sTNF-R). The results were compared with blood-CSF barrier function, cell count and the intrathecal synthesis of HTLV-I antibodies. No correlation could be demonstrated. High levels of sTNF-R were found in CSF of patients with HTLV-I-associated myelopathy and multiple sclerosis. In addition, intrathecal sTNF-R was also detected in the patients with non-inflammatory diseases, indicating that sTNF-R is definitively a normal constituent of CSF.

Vaccination of rabbits with recombinant vaccinia virus carrying the envelope gene of human T-cell lymphotropic virus type I.

Two groups of 3 rabbits each were immunized with either recombinant vaccinia virus, WR-SFB5env, carrying the human T-cell lymphotropic virus type I (HTLV-I) env gene at the site of the hemagglutinin gene of the WR strain, or control vaccinia virus, HA-WR, lacking the functional hemagglutinin gene. All 6 rabbits responded with anti-vaccinia virus antibodies. WR-SFB5env elicited anti-HTLV-I env antibodies but no vesicular stomatitis virus (HTLV-I) pseudotype neutralizing antibodies in all 3 rabbits. After 10 weeks, the animals were challenged by transfusion of blood from an HTLV-I-infected rabbit. Two of the 3 vaccinated rabbits and all 3 control rabbits became infected with HTLV-I, as indicated by seroconversion and detection of HTLV-I proviral sequences by polymerase chain reaction. The rabbit that had been protected from initial challenge became infected with HTLV-I upon rechallenge 12 weeks after the first challenge. In view of the proven prophylactic effect of passive immunization against HTLV-I, our vaccine trial failed because WR-SFB5env was incapable of inducing neutralizing antibodies against HTLV-I in the immunized animals. It remains to be studied whether cell-mediated immunity such as antibody-dependent cellular cytotoxicity was involved in the temporary protection of I vaccinated rabbit.

Comparative analysis of the antibody response to the HTLV-I gag and env proteins in HTLV-I asymptomatic carriers and HAM/TSP patients: an isotype and subclass analysis.

We have compared the immunoglobulin isotype and IgG subclass and the titre of neutralizing antibody responses to the human T cell lymphotropic virus type I (HTLV-I) between a group of asymptomatic HTLV-I infected individuals and a group with the neurological disease HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). A western blot titration assay and an envelope peptide ELISA were used to determine the presence and titre of isotype and IgG subclass responses to the gag p19 and p24 proteins and to the envelope protein. Significant increases were observed in the number of individuals seropositive for a particular isotype and IgG subclass in the HAM/TSP group versus the asymptomatic group particularly for IgM and IgE and to a lesser extent, IgA. The predominant IgG subclasses to the HTLV-I p19, p24 and envelope proteins were IgG1 and IgG3. This finding was also observed in the titres of the antibody responses to these HTLV-I proteins. The HAM/TSP group also exhibited significantly higher neutralizing antibody titres than the asymptomatic group. This evidence suggests that some form of chronic immune stimulation might be involved in the immunopathogenesis of HAM/TSP. In addition, by following the Western blot titre to the IgM and IgE isotypes in particular, it may be possible to identify asymptomatic individuals progressing to HAM/TSP.

Infection of rats with human T-cell leukemia virus type-I: susceptibility of inbred strains, antibody response and provirus location.

The susceptibilities of different strains of inbred rats to infection with the human T-cell leukemia virus (HTLV-I) after inoculation of human HTLV-I producer cell lines were compared. The Fisher F344 and Brown Norway strains developed the highest antibody response to HTLV-I, while the Lewis and BB strains were low responders. Antibodies against the HTLV-I gag proteins, and env gp21 but not env gp46, were detected in Western blots with sera from HTLV-I-infected Fischer F344 and Brown Norway rats. These sera were inactive in an in vitro syncytium-formation inhibition test. The HTLV-I provirus was detected by polymerase chain reaction in all Fischer F344, and some Lewis and Brown Norway rats, but not in the BB, which lack CD8+ T lymphocytes. The most frequent locations of the HTLV-I provirus in the Fischer F344, Lewis and Brown Norway rats at 12 weeks after infection were the peripheral blood mononuclear cells (PBMC) and spinal cord. In a second experiment in Brown Norway rats, the provirus was again detected in the PBMC of rats at 12 weeks, but not at 22 weeks, and among the other organs tested at 22 weeks the sympathetic nerve ganglia were positive. It is concluded that HTLV-I infection occurs in adult rats, but is suppressed with time.

Viral specific humoral immune responses following transfusion-related transmission of human T cell lymphotropic virus type-I infection

The immunoglobulin (Ig) isotypes of antibodies to specific proteins of the human T cell lymphotropic virus type I (HTLV-I) were determined by Western blot analysis of serial specimens from six individuals who experienced HTLV-I seroconversion following blood transfusion; five remained asymptomatic carriers, while one developed HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) 32 weeks posttransfusion. Analysis of Ig isotypes demonstrated that while IgM was the most frequent early response to gag (p19, p24) and env (r21e) proteins within the first 3 months following transfusion, IgG and IgA responses could also be detected within this period. HTLV-I-specific antibody responses plateaued in all Ig isotypes, including IgM, wtihin the next 4- to 6-months period following transfusion and pesisted through the entire study period (> 4 years). Comparison of antibody profiles in Ig isotypes and IgG1 and IgG3 subclass among asymptomatic carriers and one individual who developed HAM/TSP demonstrated no evidence of isotypic prominence or IgG subclass restriction in either group. These results indicate the appearance of HTLV-I-specific IgM that persists even after the primary infection and suggest that such responses does not appear to provide an early marker of seroconversion. Further, we found no evidence of isotypic prominence or restriction of the antibody response in recipients who remained asymptomatic compared to one who developed HAM/TS.(AU)

HTLV-1-associated myelopathy/tropical spastic paraparesis-like rats by intravenous injection of HTLV-1-producing rabbit or human T-cell line into adult WKA rats.

We intravenously injected Ra-1 cells or MT-2 cells into female adult WKA rats. Spastic paraparesis mainly in the hind-limbs was observed in 1 out of 2 Ra-1 cell-injected WKA rats and in 3 out of 8 MT-2 cell-injected WKA rats 20-27 months after injection. The main neuropathological finding was symmetrical white matter degeneration with mononuclear cell infiltration of the spinal cord, similar to that of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, and degeneration of nerve roots and peripheral nerves. Antibodies against HTLV-1 antigens were detected in plasma and cerebrospinal fluid from these HAM/TSP-like rats. HTLV-1 provirus was detected from the peripheral blood mononuclear cells of one of these rats 20 months after injection. Interestingly, spastic paraparesis was not observed in F344 rats.

Antibody responses to the env epitopes of human T-lymphotropic virus type I in rhesus macaques' naturally infected with simian T-lymphotropic virus type I.

Synthetic peptides derived from the env protein of human T-lymphotropic virus type I (HTLV-I) were used to identify the immunodominant motifs in rhesus macaques naturally infected with simian T-lymphotropic virus type I (STLV-I). Of the 13 peptides derived from the env protein of HTLV-I, Env-1(191-214) and Env-5(242-257) reacted with 81% (44/54) and 54% (29/54) of specimens from infected monkeys, respectively. A recombinant protein (MTA-I162-209) reacted with 53 of 54 (98%) STLV-I-infected serum specimens. While similar immune reactivities to Env-1 and MTA-1 were observed in both HTLV-I-infected human sera and STLV-I-infected monkey sera, the reactivity to Env-5 was significantly higher in HTLV-I-infected human sera. Differential reactivities may be attributed to the differences in the secondary structures of the STLV-I and HTLV-I envelope protein in the Env-5 region, since homologous peptide with STLV-I sequence did not result in enhanced sensitivity of anti-Env-5 antibody detection.

Characterization of the antibody response to three different versions of the HTLV-I envelope protein expressed by recombinant vaccinia viruses: induction of neutralizing antibody.

Recombinant vaccinia viruses (RVV) designated RVV E1, RVV E2, and RVV E3, were constructed to express three different versions of the human T cell leukemia virus type I (HTLV-I) envelope proteins to determine which configuration elicits an optimal antibody response. RVV E1 expressed the native HTLV-I envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while RVV E2 expressed the envelope precursor with the proteolytic cleavage site deleted. The RVV E3 construct expressed only the external surface glycoprotein, gp46. Radioimmunoprecipitation and FACS analysis confirmed that the appropriate envelope proteins were expressed by RVV E1-, E2-, and E3-infected cells. Immunization studies were carried out using Balb/c, A/J, and C57BL/6 strains of mice. Balb/c mice responded poorly to immunization with all of the three RVV constructs. C57BL/6 mice produced neutralizing antibodies in response to immunization with all three constructs, whereas A/J mice developed neutralizing antibodies only when immunized with the RVV E1s construct. The results indicate that the humoral immune responses depend on the form of HTLV-I envelope proteins expressed by each RVV.

Comparative biological responses of rabbits infected with human T-lymphotropic virus type I isolates from patients with lymphoproliferative and neurodegenerative disease.

An experimental rabbit model was used to determine host responses to infection by various human T-lymphotropic virus type-I (HTLV-I) strains. Seven groups of 4 to 5 rabbits each were inoculated with lethally-irradiated HTLV-I-infected cell lines derived from patients with adult T-cell leukemia/lymphoma or from patients with HTLV-I-associated myelopathy. Four separate control groups of 2 rabbits each were inoculated with similarly prepared HTLV-I-negative cells derived from rabbits or humans. Anti-viral antibody responses were assessed by immunoblot assay and hematologic parameters were measured using automated cell counters and cytologic staining. The virologic status of challenged rabbits was determined by co-culture and HTLV-I antigen capture assay, as well as by polymerase chain reaction (PCR) amplification of HTLV-I DNA from peripheral blood mononuclear cells (PBMC) or tissues. The HTLV-I inocula could be separated into groups based upon their infectivity to rabbits: highly infectious strains elicited intense serologic responses and were detected frequently in tissues by antigen and PCR assays, while other strains were moderately to poorly infectious, induced weak antibody responses and were infrequently detected by antigen and PCR assays. Overall, PBMC appeared to have the greatest quantity of HTLV-I containing cells, while bone marrow was a poor source of virus. No clinical or hematologic abnormalities were evident during the 24-week course of infection. Taken together, our results suggest there is heterogeneity in the biological response to HTLV-I infection which is, in part, dependent on the infecting strain of virus.

Immunoglobulin prophylaxis against milkborne transmission of human T cell leukemia virus type I in rabbits.

Prophylactic effect of human T cell leukemia virus type I (HTLV-I) immune globulin (HTLVIG) against milkborne transmission of HTLV-I was investigated in a rabbit model. Four litters (A-D: 7, 5, 7, and 7 offspring, respectively) born to an HTLV-I-infected rabbit were used. Litters A and D were allowed to grow normally as controls, while litters B and C were given weekly intraperitoneal inoculation of HTLVIG four times until weaning at 4.5 weeks of age. Only 1 (8.3%) of the 12 HTLVIG-inoculated rabbits, compared with 6 (42.9%) of the 14 control rabbits, seroconverted for HTLV-I. Gene amplification detected the presence of HTLV-I proviral sequences in all of the seroconverted but in none of the seronegative rabbits. These results suggest that passive immunization is effective in preventing dam-to-offspring transmission of HTLV-I.

A synthetic peptide elicits antibodies reactive with the external glycoprotein of human T cell lymphotropic virus type I.

A synthetic peptide derived from the external glycoprotein of human T cell lymphotropic virus type I (HTLV-I) (Env-5; amino acids 242 to 256) reacted with IgG antibodies in serum specimens from HTLV-I-infected individuals. C-terminal residues of Env-5 were crucial for the antibody reactivity. Polyclonal rabbit antibodies to Env-5 did not inhibit syncytium formation but such antibodies reacted specifically with gp68env and gp46env glycoproteins of HTLV-I in an immunoblot analysis. Immunoprecipitation of the surface-labelled MT-2 (HTLV-I-infected) cell line with anti-Env-5 precipitated the gp68env precursor protein. It was concluded that peptide Env-5 mimics a surface-exposed epitope on the HTLV-I external glycoprotein.

Characterization of immunodominant epitopes of gag and pol gene-encoded proteins of human T-cell lymphotropic virus type I.

A series of synthetic peptides derived from the corresponding regions of the gag, pol, and env proteins of human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were used in an enzyme immunoassay to map the immunodominant epitopes of HTLV. Serum specimens from 79 of 87 (91%) HTLV-I-infected patients reacted with the synthetic peptide Gag-1a (amino acids [a.a.] 102 to 117) derived from the C terminus of the p19gag protein of HTLV-I. Minimal cross-reactivity (11%) was observed with serum specimens from HTLV-II-infected patients. Peptide Pol-3, encoded by the pol region of HTLV-I (a.a. 487 to 502), reacted with serum specimens from both HTLV-I- and HTLV-II-infected patients (94 and 86%, respectively). The antibody levels to Pol-3 were significantly higher (P less than 0.01) in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in either adult T-cell leukemia patients or HTLV-I-positive asymptomatic carriers. None of the other peptides studied demonstrated significant binding to serum specimens obtained from HTLV-I- or HTLV-II-infected individuals. While Gag-1a did not react with serum specimens from normal controls, Pol-3 demonstrated some reaction with specimens from seronegative individuals (11.4%). The antibodies to Gag-1a and Pol-3 in serum specimens from HTLV-I-infected patients could be specifically inhibited by the corresponding synthetic peptides and by a crude HTLV-I antigen preparation, indicating that these peptides mimic native epitopes present in HTLV-I proteins that are recognized by serum antibodies from HTLV-I- and -II-infected individuals.