Establishment of a novel diagnostic test algorithm for human T-cell leukemia virus type 1 infection with line immunoassay replacement of western blotting: a collaborative study for performance evaluation of diagnostic assays in Japan.

BACKGROUND: The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. RESULTS: Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. CONCLUSIONS: Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

The novel immunogenic chimeric peptide vaccine to elicit potent cellular and mucosal immune responses against HTLV-1.

This study reports on the immunogenicity assessment of a novel chimeric peptide vaccine including Tax, gp21, gp46, and gag immunodominant epitopes of human T-cell lymphotropic virus type 1 (HTLV-1) to induce immunity against HTLV-1 after subcutaneous (SC) or intranasal administration in a mice model. Additionally, to elevate the efficacy of the HTLV-1 vaccine, the chimera was physically mixed with monophosphoryl lipid A (MPLA) or ISCOMATRIX (IMX) adjuvants. For this purpose, the ISCOMATRIX with a size range of 40-60 nm were prepared using lipid film hydration method. Our investigation revealed that the mixture of IMX and chimera could significantly increase antibody titers containing IgG2a, and mucosal IgA, as well as IFN-γ and IL-10 cytokines and decrease the level of TGF-ß1, compared to other vaccine formulations. The intranasal delivery of chimera vaccine in the absence or presence adjuvants stimulated potent mucosal sIgA titer relative to subcutaneous immunization. Furthermore, the SC or nasal delivery of various vaccine formulations could shift the immunity toward cell-mediated responses, as evident by higher IgG2a and IFN-γ, as well as suppressed TGF-ß1 level. Our findings suggest that proper design, construction, and immunization of multi-epitope vaccine are essential for developing an effective HTLV-1 vaccine.

Development and Evaluation of a Novel ELISA for Detection of Antibodies against HTLV-I Using Chimeric Peptides.

We aimed to develope a peptide-based indirect ELISA to detect antibodies against Human T-lymphotropic virus type I (HTLV-I). Two chimeric peptides (CP-1 and CP-2) were designed using linear immunodominant epitopes of gp-46-I, and gp21-I proteins, according to the sequence from Uniprot database. These peptides were studied initially in the ELISA using infected sera. The most promising peptideCP-1, was used to develop a peptide ELISA for detection of HTLV-I infected sera. The optimal conditions for CP-1ELISA were: the optimum coating buffer was 100mM NaHCO3, pH 9.6; coating peptide concentration was 10 µg/mL; the optimal blocking buffer was5% fetal bovine serum (FBS); the secondary antibody concentration was 1:2000; and serum dilution was 1:20. 20serum samples from HTLV-I infected patients were evaluated by ELISA developed. CP-1 showed high antigenicity while lacking any cross-reactivity with normal human sera. The results of evaluations indicated that in comparison with commercial ELISA, CP-1 ELISA showed good sensitivity and specificity. With further validation, CP-1as described in the present study could be introduced as novel reliable and cost-effective candidates for the high-specific screening of HTLV-I/-II infections in endemic regions.

Reevaluation of confirmatory tests for human T-cell leukemia virus Type 1 using a luciferase immunoprecipitation system in blood donors.

BACKGROUND: Recently, Japanese Red Cross blood centers have changed the confirmatory test method from an indirect immunofluorescence (IF) technique to Western blotting (WB) for antibodies against human T-cell leukemia virus Type 1 (HTLV-1). In this study, these HTLV-1 tests were assessed using another sensitive method, that is, a luciferase immunoprecipitation system (LIPS), to identify a better confirmatory test for HTLV-1 infection. STUDY DESIGN AND METHODS: Plasma samples from 54 qualified donors and 114 HTLV-1 screening-positive donors were tested by LIPS for antibodies against HTLV-1 Gag, Tax, Env, and HBZ recombinant proteins. The donors were categorized into six groups, namely, (Group I) qualified donors, screening positive; (Group II) IF positive; (Group III) IF negative; (Group IV) WB positive; (Group V) WB negative; and (Group VI) screening positive in the previous blood donation, but WB-indeterminate during this study period. RESULTS: In Groups II and IV, all plasma samples tested positive by LIPS for antibodies against Gag and Env proteins. In Group V, all samples tested negative by LIPS, whereas some Group III samples reacted with single or double antigens in LIPS. In Group VI, the LIPS test identified a donor with suspected HTLV-1 infection. The first case of a blood donor with plasma that reacted with HBZ was identified by LIPS. CONCLUSION: Reevaluation of the current HTLV-1 screening method using the LIPS test showed that both confirmatory tests had similar sensitivity and specificity only when WB indeterminate results were eliminated. LIPS is a promising method for detecting and characterizing HTLV-1 antibodies.

HTLV-1 bZIP factor-specific CD4 T cell responses in adult T cell leukemia/lymphoma patients after allogeneic hematopoietic stem cell transplantation.

We document human T lymphotropic virus type 1 (HTLV-1) bZIP factor (HBZ)-specific CD4 T cell responses in an adult T cell leukemia/lymphoma (ATL) patient after allogeneic hematopoietic stem cell transplantation (HCT) and identified a novel HLA-DRB1*15:01-restricted HBZ-derived naturally presented minimum epitope sequence, RRRAEKKAADVA (HBZ114-125). This peptide was also presented on HLA-DRB1*15:02, recognized by CD4 T cells. Notably, HBZ-specific CD4 T cell responses were only observed in ATL patients after allogeneic HCT (4 of 9 patients) and not in nontransplanted ATL patients (0 of 10 patients) or in asymptomatic HTLV-1 carriers (0 of 10 carriers). In addition, in one acute-type patient, HBZ-specific CD4 T cell responses were absent in complete remission before HCT, but they became detectable after allogeneic HCT. We surmise that HTLV-1 transmission from mothers to infants through breast milk in early life induces tolerance to HBZ and results in insufficient HBZ-specific T cell responses in HTLV-1 asymptomatic carriers or ATL patients. In contrast, after allogeneic HCT, the reconstituted immune system from donor-derived cells can recognize virus protein HBZ as foreign, and HBZ-specific immune responses are provoked that contribute to the graft-versus-HTLV-1 effect.

How does HTLV-I persist despite a strong cell-mediated immune response?

Human T-lymphotropic virus type 1 (HTLV-1) is a pathogenic retrovirus that infects human CD4(+) T lymphocytes. Despite its presence in T cells, HTLV-1 causes little overt immunosuppression. This host-virus relationship has therefore been exploited as an excellent model system for studying the dynamic interaction between a persistent retrovirus and the normal human immune system. We use a combination of mathematical and experimental techniques to identify key factors on both sides of the in vivo host-virus interaction that significantly determine HTLV-I proviral load and disease risk. We develop a model to describe how these factors interact to enable viral persistence.

Comparison of four commercial screening assays for the diagnosis of human T-cell lymphotropic virus types 1 and 2.

Serological assays for human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) are widely used in routine screening of blood donors. The aim of this study was to compare the performance of four commercial screening assays for HTLV-1/2 infection frequently used in South America. A total of 142 HTLV-1 and HTLV-2 seropositive and 336 seronegative samples were analyzed by using four commercial tests (BioKit, Vironostika, Murex and Fujirebio). These tests are commonly used for HTLV-1/2 detection in blood banks in Argentina. A nested-PCR was used as the reference standard. The most sensitive tests for HTLV-1/2 were Fujirebio and Biokit (98.6%) followed by Murex (97.2%) and Vironostika (96.5%). The most specific test was Murex (99.7%), followed by Biokit (97.0%), Fujirebio (95.8%), and Vironostika (92.9%). The kappa index of agreement was higher for Murex (kappa=0.97), followed by BioKit (kappa=0.94), Fujirebio (kappa=0.92), and Vironostika (kappa=0.86). The highest index of agreement was shown by Murex test while Vironostika had the lowest performance. Of the four tests evaluated, only the Vironostika assay is approved by the Food and Drug Administration. These results should be considered for choosing the most accurate serological screening assays in order to obtain an optimal efficiency of the current algorithm for HTLV-1/2 diagnosis.

Antibody to the human T-lymphotropic virus type 1 (HTLV-1) envelope protein Gp46 in patients co-infected with HCV and HTLV-1.

Human T-lymphotropic virus type 1 (HTLV-1) infection is known to affect hepatitis C virus (HCV) clearance and to accelerate the development of hepatocellular carcinoma in HCV-infected patients. In this study, we found the prevalence and titer of an antibody recognizing the central region of the HTLV-1 Gp46 protein to be associated with the severity of chronic liver disease. The antibody prevalence was significantly correlated with the stage of chronic liver disease (P < 0.0001): 3 (14.3%) of 21 patients with minimal-mild chronic hepatitis, 12 (24%) of 50 with moderate-severe chronic hepatitis, 7 (87.5%) of 8 with liver cirrhosis, and 13 (100%) of 13 with hepatocellular carcinoma. These results indicate that the antibody may be a useful marker of the deterioration of liver disease in patients co-infected with HCV and HTLV-1. This antibody may be useful for the diagnosis of liver diseases and the development of more effective treatments.

Ex vivo analysis of human T lymphotropic virus type 1-specific CD4+ cells by use of a major histocompatibility complex class II tetramer composed of a neurological disease-susceptibility allele and its immunodominant peptide.

HLA-DRB1*0101 is associated with susceptibility to human T lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Here, we used a synthetic tetramer of DRB1*0101 and its epitope peptide to analyze HTLV-1-specific CD4(+) T cells ex vivo. The frequency of tetramer(+)CD4(+) T cells was significantly greater in patients with HAM/TSP than in healthy HTLV-1 carriers (HCs) at a given proviral load and correlated with HTLV-1 tax messenger RNA expression in HCs but not in patients with HAM/TSP. These cells displayed an early to intermediate effector memory phenotype and were preferentially infected by HTLV-1. T cell receptor gene analyses of 2 unrelated DRB1*0101-positive patients with HAM/TSP showed similar Vbeta repertoires and amino acid motifs in complementarity-determining region 3. Our data suggest that efficient clonal expansion of virus-specific CD4(+) T cells in patients with HAM/TSP does not simply reflect higher viral burden but rather reflects a rapid turnover caused by preferential infection and/or in vivo stimulation by major histocompatibility complex-peptide complexes.

Seroprevalence and molecular epidemiology of human T-Cell leukemia virus type 1 (HTLV-1) and HTLV-2 in blood donors from Dakar, Senegal.

In 2002, human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 seroprevalence was 0.16% (8/4,900) in blood donors from Dakar, Senegal. Most of the positive donors originated from the country's southern region. Seven donors were infected by HTLV-1 (of cosmopolitan subtype), and one was infected by HTLV-2. These data highlight the problem of transfusion safety in this area where HTLV-1-associated lymphoproliferative and neurological diseases are endemic.

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

Human leukocyte antigen-class II-negative long-term cultured human T-cell leukemia virus type-I-infected T-cell lines with progressed cytological properties significantly induce superantigen-dependent normal T-cell proliferation.

While most human T-cell leukemia virus type-I (HTLV-I)-infected T cells express abundant class II antigens, some aggressive-type adult T-cell leukemia (ATL) cells lose their expression. To investigate the significance of the class II antigen of HTLV-I infected cells, the progressiveness of HTLV-I-infected long-term cultured T-cell lines was evaluated, and then their antigen-presenting capacity was examined using a superantigen, staphylococcus enterotoxin B (SEB). Among the cell lines derived from peripheral blood, HPB-ATL-T (ATL-T), HPB-ATL-2 (ATL-2) and HPB-ATL-O were more progressed than Tax exclusively expressing HPB-CTL-I (CTL-I), because the former deleted p16 gene (polymerase chain reaction (PCR)) and strongly transcribed survivin (reverse transcriptase-PCR). Notably, interferon gamma-independent loss of class II expression of ATL-T and ATL-2 was found. In antigen-presenting experiments, however, both cell lines induced SEB-dependent significant T-cell proliferation estimated by [(3)H] thymidine uptake. No class II-re-expressed ATL-2 cells were observed in the SEB-presenting cultures by indirect immunofluorescence, and only minimum inhibition of SEB-dependent T-cell response by anti-human leukocyte antigen (HLA)-DR monoclonal antibody was observed. These findings suggest that both ATL-T and ATL-2 very effectively present SEB to T cells less dependently on class II molecules. These less immunogenic leukemic cells of aggressive ATL may contribute to disease aggression.

Antigenic mixture of synthetic peptides for the immunodiagnosis of HTLV I/II infection.

Monomeric and chimeric synthetic peptides were used as coating antigens in four different mixtures in a solid phase immunoassay to select an optimal combination for the detection of antibodies to human T-cell lymphotropic virus (HTLV) in serum samples. The peptides, P-13 (gp21 I), Q5 (gp21 II)-GG-(gp46 II), and Q (gp46 I)-GG-(p19 I), represent immunodominant sequences from transmembrane protein (gp21), envelope protein (gp46), and core protein (p19) of HTLV I/II viruses; they were the most antigenic and specific peptides in previous studies. The sequences of the chimeric peptides were separated by two glycine residues. An indirect UltramicroEnzyme-linked immunosorbent assay (UMELISA) was used to evaluate the antigenicity of these peptide mixtures by using samples from anti-HTLV I/II PRP205(M), (n = 20), HTLV I-infected individuals from Cuba (n = 7), and HTLV I-positive sera from Colombia and Chile (n = 9). The specificity was evaluated with healthy blood donor sera (n = 300), anti-HIV 1-positive samples (n = 10), and other seropositive samples to different infectious agents. The highest sensitivity and specificity was obtained with mixture 1, which could be very useful in the immunodiagnostic of HTLV infection.

[Sero-epidemiological study on the human T-cell leukaemia virus type I/II infection in the east coastal areas of Fujian province].

OBJECTIVE: To study the seroprevalence of human T-cell leukaemia virus type I/II (HTLV-I/II) infection in adult population in the east coastal areas of Fujian and to explore the possible risk factors of HTLV-I/II. METHODS: A total number of 3259 blood samples from drug users, sexually transmitted disease (STD) patients, prostitutes and blood donors for serologic assays during 1999 to 2002, were collected. All samples were screened for HTLV-I/II antibody, using enzyme linked immunosorbent assay (ELISA) kits. All of the positive samples were confirmed by western blot (WB) kits. Statistical analysis was done by Epi software, and chi(2) test by Fisher's exact test. P value < 0.05 was considered statistically significant. RESULTS: The overall seroprevalence rate of HTLV-I/II in healthy populations was 0.06% including, 0.32% in drug users, 0.58% in STD patients and prostitutes respectively. HTLV-II had not been found. The seropositive rates for HTLV-I in STD patients and prostitutes were significantly higher than the findings among healthy populations (P < 0.05). There were no different seroprevalence rates between drug users and healthy populations (P > 0.05). No significant changes in HTLV-I prevalence rates were found in the different age groups as well as in Fuzhou and Linde cities (P > 0.05). CONCLUSION: The result suggested that in the east coastal areas of Fujian province, HTLV-I was the main prevalent virus. The seroprevalence of HTLV-I was very low, with no HTLV-II. Neither age nor gender seemed to be HTLV-I risk factor in the east coastal areas of Fujian province, but the increase of exposure to sex might be one.

Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.

T cells accumulating in the inflamed joints of a spontaneous murine model of rheumatoid arthritis become restricted to common clonotypes during disease progression.

Although a number of studies have revealed that T cells expand clonally in the joints of patients suffering from rheumatoid arthritis (RA), the kinetics of T cell clonality in multiple joints of an individual throughout progression of the disease is not known. By employing a TCR beta chain gene-specific RT-PCR and subsequent single-strand conformation polymorphism, which enables us to monitor T cell clonality, we analyzed transgenic mice (Tg) carrying the human T cell leukemia virus type I env-pX region. These mice spontaneously develop destructive progressive arthritis similar to RA as they age. In the early stage, the majority of accumulating T cell clones differed in each of four affected feet analyzed. However, in the advanced stage, many of the clones were common to all four feet. The total number of distinct clones gradually decreased as the disease progressed. When splenocytes from arthritic elder Tg were adoptively transferred into either nude mice or young Tg, the clones common to all four feet of the donor were detected again in four feet of the recipients. These findings suggest that, as arthritis progresses, the T cell clones accumulating in the arthritic joints are gradually restricted to certain common clonotypes, some of which are arthrotropic.

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.

Expansion of human T-cell leukemia virus type 1 (HTLV-1) reservoir in orally infected rats: inverse correlation with HTLV-1-specific cellular immune response.

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals. Although the critical risk factor for ATL development is not clear, it has been noted that ATL is incidentally associated with mother-to-child infection, elevated proviral loads, and weakness in HTLV-1-specific T-cell immune responses. In the present study, using a rat system, we investigated the relationships among the following conditions: primary HTLV-1 infection, a persistent HTLV-1 load, and host HTLV-1-specific immunity. We found that the persistent HTLV-1 load in orally infected rats was significantly greater than that in intraperitoneally infected rats. Even after inoculation with only 50 infected cells, a persistent viral load built up to considerable levels in some orally infected rats but not in intraperitoneally infected rats. In contrast, HTLV-1-specific cellular immune responses were markedly impaired in orally infected rats. As a result, a persistent viral load was inversely correlated with levels of virus-specific T-cell responses in these rats. Otherwise very weak HTLV-1-specific cellular immune responses in orally infected rats were markedly augmented after subcutaneous reimmunization with infected syngeneic rat cells. These findings suggest that HTLV-1-specific immune unresponsiveness associated with oral HTLV-1 infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, but could be overcome with immunological strategies.

Degenerate specificity of HTLV-1-specific CD8+ T cells during viral replication in patients with HTLV-1-associated myelopathy (HAM/TSP).

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-1 infection, in which HTLV-1-infected CD4(+) T cells and HTLV-1-specific CD8(+) T cells may play a role in the disease pathogenesis. Patients with HAM/TSP have high proviral loads despite vigorous virus-specific CD8(+) T-cell responses; however, it is unknown whether the T cells are efficient in eliminating the virus in vivo. To define the dynamics of HTLV-1-specific CD8(+) T-cell responses, we investigated longitudinal alterations in HTLV-1 proviral load, amino acid changes in an immunodominant viral epitope, frequency of HTLV-1-specific T cells, and degeneracy of T-cell recognition in patients with HAM/TSP. We showed that the frequency and the degeneracy of the HTLV-1-specific CD8(+) T cells correlated well with proviral load in the longitudinal study. The proviral load was much higher in a patient with low degeneracy of HTLV-1-specific T cells compared to that in a patient with comparable frequency but higher degeneracy of the T cells. Furthermore, in a larger number of patients divided into 2 groups by the proviral load, those with high proviral load had lower degeneracy of T-cell recognition than those with low proviral load. Sequencing analysis revealed that epitope mutations were remarkably increased in a patient when the frequency and the degeneracy were at the lowest. These data suggest that HTLV-1-specific CD8(+) T cells with degenerate specificity are increased during viral replication and control the viral infection.

Sequence variations in the amino- and carboxy-terminal parts of the surface envelope glycoprotein of HTLV type 1 induce specific neutralizing antibodies.

The surface envelope glycoprotein gp46 of the human T cell leukemia virus type 1 elicits a strong immune response. Its protective role against HTLV-1 infection in animal models is well established, suggesting that recombinant envelope glycoproteins or synthetic peptides could be used as an effective vaccine. However, reports have indicated that some variations in envelope sequences may induce incomplete cross-neutralization between HTLV-1 strains. To identify amino acid changes that might be involved in induction of specific neutralizing antibodies, we studied sera from three patients (2085, 2555, and 2709) infected by HTLV-1 with surface glycoprotein gp46 harboring variations in amino acid sequence at positions 39, 72, 265, and 290. Inhibition of syncytia induced by parental, chimeric, or point-mutated envelope proteins indicated that sera 2555 and 2709 primarily recognized neutralizable epitopes located in N- and C-terminal parts of the gp46 glycoprotein. Amino acids changes at positions 39, 265, and 290 greatly impaired recognition of neutralizing epitopes recognized by these two sera. These results demonstrate that amino acid changes in envelope glycoprotein gp46 can induce strain-specific neutralizing antibodies in some patients. On the other hand, the neutralizing activity of serum 2085 was not affected by amino acid changes at positions 39, 265, and 290, suggesting that the neutralizing antibodies present in this serum were directed against epitopes located in other parts of the molecule, possibly those located in the central domain of the molecule, which has the same amino acid sequence in the three viruses.