Association between maternal antibodies to the external envelope glycoprotein and vertical transmission of human T-lymphotropic virus type I. Maternal anti-env antibodies correlate with protection in non-breast-fed children.

Vertical transmission of human T-lymphotropic virus type I (HTLV-I) depends primarily on breast-feeding; substitution of bottle-feeding has reduced the transmission rate from 20% in breast-fed children to 3% among bottle-fed. To determine the correlates of transmission for long breast-feeding (> or = 6 mo), short breast-feeding (< 6 mo), and bottle-feeding mothers, the antibody titers of transmitter (T) mothers and non-transmitter (nT) mothers were analyzed by using synthetic and recombinant epitopes representing the immunodominant epitopes of gag (Gag1a, r24), env (Env1/5, MTA1, RE3), and tax (Tax8/22-24) proteins. Seroreactivity to gag and tax epitopes was not significantly different except for anti-r24 antibody titer, which was significantly higher among T-mothers (geometric mean 134) when compared with nT-mothers (62) in the long-feeding group (P < 0.001). Profiles of antibody titers against env epitopes were different. Within the long-feeding group, Env1/5, MTA1, and RE3 titers were significantly higher among T-mothers (258, 1,476, and 738, respectively) when compared with nT-mothers (106, 279, and 320, respectively) (P < 0.01 for all three epitopes). In contrast, within the bottle-feeding group, antibody titers to Env1/5 (269) and RE3 (418) among nT-mothers were significantly higher than those among T-mothers (80 and 113, respectively) (P < 0.01). These data confirm that high-titered anti-HTLV-I antibodies in the long-feeding group correlate with milk-borne transmission of HTLV-I and, more importantly, imply that maternal anti-env antibodies may reduce the risk of non-milkborne infection.

Proposal for diagnostic criteria of tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).

After the first description of TSP/HAM in 1985 and the elaboration of WHO's diagnostic criteria in 1988, the experience of the professionals in this field has increased so that a critical reappraisal of these diagnostic guidelines was considered timely. Brazilian neurologists and observers from other countries met recently to discuss and propose a modified model for diagnosing TSP/HAM with levels of ascertainment as definite, probable, and possible, according to myelopathic symptoms, serological findings, and/or detection of HTLV-I DNA and exclusion of other disorders.

Reflux of HTLV-I infected lymphocytes from the privileged compartment(s) to peripheral blood flow in patients with HTLV-I-associated myelopathy.

To understand the mechanisms involved in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), three in vivo phenomena which have been observed in the peripheral blood of patients and differing from that in asymptomatic HTLV-I carriers must be taken into consideration: (a) the presence of increased HTLV-I viral load, (b) a higher immune responsiveness against HTLV-I antigens, and (c) biased nucleotide substitutions in the HTLV-I pX region which indicate a decreased selection pressure for viral amino acid changes. We now propose a hypothesis which focuses on the in vivo dynamics of HTLV-I infected lymphocyte migration and which incorporates these features. In addition, the hypothesis assumes the existence of a deviation in immune surveillance for HTLV-I in the central nervous system (CNS) in spite of the presence of frequent specific immune effectors. We suggest that in the active phase of HAM/TSP, accompanied with or following autoaggressive interactions between infected lymphocytes and immunocompetent cells in the CNS, there is a consequential reflux of the infected lymphocytes to the peripheral blood. The reflux of infected cells would be expected to provide peripheral blood with tissue-derived HTLV-I proviruses which have been indulged and propagated in an immune-privileged site. This process would result in and account for the observed increase in viral load and the substitution bias in HTLV-I sequences in the peripheral blood.

Humoral responses to the immunodominant gag and env epitopes of human T-lymphotropic virus type I among Melanesians.

The immune responsiveness to the immunodominant B-cell epitopes of human T-lymphotropic virus type I (HTLV-I), derived from the external envelope glycoprotein (recombinant MTA-1(162-209), synthetic Env-1(194-214), and Env-5(242-257)) and the gag-encoded matrix protein (Gag-1a(102-117)), was analyzed in 19 HTLV-I-seropositive and 51 HTLV I-seroindeterminate Melanesians from Papua New Guinea and the Solomon Islands. The reactivities of seropositive Melanesian specimens to MTA-1 (100%), Env-5 (89%), and Gag-1a (79%) were similar to that seen with U.S. specimens, while reactivity to Env-1 was lower in Melanesian specimens (68%). Minimal reactivity was found to the env epitopes among the 51 HTLV-I-seroindeterminate Melanesians, but 29 (57%) reacted to Gag-1a. The failure to detect HTLV-I gag, pol, env, and tax gene sequences by polymerase chain reaction among the seroindeterminate Melanesians suggests that such reactivities to the Gag-1a epitope represent cross-reacting antibodies with closely related microbial or cellular proteins.

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, within the next 4- to 6-month period following transfusion and persisted 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 response 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/TSP.

Influence of amino acid substitutions on antigenicity of immunodominant regions of the HTLV type I envelope surface gylcoprotein: a study using monoclonal antibodies raised against relevant peptides.

By the use of sera of human T cell leukemia virus type I (HTVL-I)-infected individuals it was shown that amino acid substitutions at positions 192 (proline to serine) and 250 (serine to proline) in major immunodominant regions (175-199 and 239-261) of the surface envelope glycoprotein (gp46) of the virus may influence the humoral response. Since human sera are polyclonal in nature, one cannot readily discriminate between an immunoglobulin-specific recognition and multiple bindings of diverse antibodies. To overcome this difficulty we generated murine monoclonal antibodies to synthetic peptides mimicking all or portions of these gp46 regions. The reactivity of some of these antibodies to synthetic peptides harboring (or not harboring) the preceding amino acid substitutions at position 192 or 250, to denatured gp46 by Western blotting, and to live (variously substituted) HTLV-I-infected cells, combined with blocking experiments with various peptides, allow us to conclude that the major epitopes (positions 183-191, 190-197, 190-199, and 246-252) in the two immunodominant regions may elicit different antibody responses according to their sequences. It is worth noting that in a reporter gene inhibition assay, it was found that a neutralizing monoclonal antibody (MF1), the epitope for which is located between residues 190 and 197, had a high level of activity when cells (2060) harboring a gp46 with proline at position 192 were used and had no activity toward cells (1010) with a serine at this position. Therefore our results establish that certain amino acid substitutions of gp46 may drastically affect the antigenicity of the molecule and the biological activity of the antibodies elicited.

HTLV-I and HTLV-II infections in volunteer blood donors and high-risk groups in Lebanon.

A serosurvey for Human T-cell Lymphotropic virus type 1 (HTLV-I)/HTLV-II was conducted in 1,900 blood donors, 120 pregnant women and 436 high-risk group patients in Beirut, Lebanon. One of the 1,900 blood donors was anti-HTLV-I/II-seroreactive on screening by enzyme immunoassay (EIA) but was indeterminate by Western blot and negative by polymerase chain reaction. None of the other 556 subjects studied was seroreactive by EIA. The credibility of the zero prevalence of HTLV-I/II infection among the Lebanese blood donors is supported by the absence of seroreactivity of antibodies in the multiply transfused patients. It seems therefore that the prevalence of HTLV-I/II appears to be less than 1 in 2,456 in the Lebanese population and hence, HTLV-I/II infection does not appear to require routine screening in Lebanon.

Production and characterization of a monoclonal antibody directed against HTLV-1 p19: use in a specific capture enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for detection of Human T-cell Leukemia Virus antigen in culture supernatants and cell lysates. The assay used a mouse monoclonal antibody against HTLV-I p19 major core protein as capture antibody. It has a sensitivity of 1 microgram/ml of HTLV-I protein, 250 pg/ml of purified recombinant p19 and detected p19 in an 10(-2) diluted supernatant of MT2 infected cell and in a 100 MT2 cells lysate (10(6) cells taken at day 7 of culture). The assay enable us to discriminate between HTLV-I and HTLV-II antigens and is reproducibly negative for supernatants and cell lysates of uninfected cells and of HIV-1 infected cells. The assay was found to be more specific and 10 times more sensitive than the reverse transcriptase (RT) assay, and the EIA test became positive three days earlier than RT assay for the HTLV-I cell lines supernatants.

Prognosis of HTLV-1 positive renal transplant recipients in Iran.

The human T lymphocyte virus-1 (HTLV-1) is the responsible pathogen for diseases such as HTLV-1 associated myelopathy (HAM) and adult T-cell leukemia (ATL). Mashhad, in northeast Iran, with high instances of this infection, has a noticeable number of infected renal failure patients. Since immunosuppressive drugs might decrease the latency period of HTLV-1 or increase its complications, the question arises whether HTLV-1 positive renal failure patients are suitable candidates for kidney transplants. To answer this, HTLV-1 positive recipients were evaluated in our study. Patients were divided into two groups. First group consisted of patients at the Imam Reza Hospital dialysis center. Second group had 20 kidney transplantation recipients consisting of ten infected and ten uninfected recipients as control from Imam Reza. Medical history of these patients was recorded and evaluated. The follow-up periods were between one and six years. Among them, 3.8% of patients undergoing dialysis were infected. The most important fact resulting from this study is that none of the infected recipients suffered from HAM or ATL during the follow-up period. In addition, it did not show any significant difference in the incidence of post-transplant complications between the infected and non-infected groups. Our study indicates that HTLV-1 positive patients may undergo kidney transplant without fear of increased incidence of side effects than those found in uninfected recipients. Because of short-term follow-up, probable long latency period of the virus, and the limited number of infected recipients, further work on this issue would be prudent.

Lack of exposure to HTLV1 among Ethiopian immigrants of operation Solomon (1991) arriving to the Jerusalem area.

HTLV1 antibodies were tested in a group of 740 Ethiopian immigrants who arrived in Israel in Operation Solomon, 1991. HTLV1 antibodies were not found in a single individual. This survey and previous ones conducted on 314 immigrants of the 1984-85 Operation Moses suggest that HTLV1 infection is not present among Ethiopian immigrants residing in Israel.

Prevalence of antibodies to HTLV in antenatal clinic attenders in south east London.

The prevalence of antibodies to HTLV in women attending a south east London antenatal clinic between October 1990 and July 1992 was determined using sera referred for routine rubella antibody testing. Samples were screened for HTLV antibody using a modified Fujirebio gel particle agglutination test and reactive sera confirmed by ELISA (Abbott Laboratories, North Chicago, IL) and two commercial Western blots (Cambridge Biotech Inc., Rockville, MD, and Diagnostic Biotechnology, Genelab Diagnostics, Louvaine, Belgium). This strategy confirmed the presence of HTLV-1 antibodies in 12 out of 6,289 sera (0.19%, 95% confidence limits 0.083% to 0.30%) and HTLV-2 antibodies in 2 (0.03%) sera. Specimens from 8 of 821 (0.97%, 95% confidence limits 0.42% to 1.9%) Afro-Caribbean women, three of 1,136 (0.26%, 95% confidence limits 0.055% to 0.78%) African women, and one of 3,049 (0.033%, 95% confidence limits 0.006% to 0.18%) Caucasian women were positive for HTLV-1 antibodies. Sera from Afro-Caribbean women born in the Caribbean were 7.6 times more likely to be HTLV-1 antibody positive than sera from Afro-Caribbean women born in the UK (P = 0.012). Selective testing of Afro-Caribbean and African antenatal clinic attenders, in this setting, would have identified 11 of the 12 HTLV-1 infections at an estimated cost of prevention of HTLV-1 associated disease of 100,000 pounds per case which is considerably less than the 1.3 million pounds which has been estimated to prevent a case by universal screening of UK blood donors.

The HTLV receptor is a widely expressed protein.

The receptor for human T-cell leukemia virus type 1 (HTLV-1) was found to be expressed on a broad range of cell lines derived from multiple species. Receptor expression was assessed using human immunodeficiency virus type 1 particles, pseudotyped with the HTLV-1 envelope glycoprotein, and expressing luciferase under the control of an SV40 enhancer and promoter. Infection by pseudotyped virus was blocked with neutralizing antibodies to HTLV-1, and infection was dependent on the presence of the cleavage and fusogenic sequences in the envelope protein precursor. Trypsin treatment of susceptible target lymphocytes reduced entry. Entry was partially resistant to ammonium chloride.

Human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, encodes a 28-kDa protein: a possible autoantigen for HTLV-I gag-reactive autoantibodies.

The presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, in the human genome has been documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames. Antibodies 232 and 233, specific for synthetic peptides pep14-24 and pep117-127, corresponding to two nonoverlapping HTLV-related regions in the longer open reading frame of HRES-1, recognize an identical 28-kDa protein in H9 human T cells. Thus, HRES-1 is a human endogenous retroviral sequence capable of protein expression. HRES-1/p28 is localized to the cytoplasm and nuclear bodies. While HTLV-I-specific antibodies react with HRES-1 peptides, antibody 233 cross-reacts with HTLV-I gag p24 protein. Three consecutive highly charged amino acid residues, Arg-Arg-Glu, present in both HRES-1 pep117-127 and HTLV-I gag p24 are likely to be the core of cross-reactive epitopes. The prevalence of antibodies to HRES-1 peptides pep14-24 and pep117-127 was determined in 65 normal blood donors and 146 patients with immunological disorders. Sera of patients with multiple sclerosis (19 out of 65, 29%), progressive systemic sclerosis (4 out of 17, 23%), systemic lupus erythematosus (4 out of 19, 21%), and Sjogren syndrome (2 out of 19, 10%) contained significantly higher HRES-1 peptide binding activity than sera of normal donors. Sera of patients with AIDS showed no specific binding to HRES-1 peptides. Nine of 30 HRES-1-seropositive patients showed immunoreactivity to HTLV-I gag p24. The data indicate that HRES-1/p28 may serve as an autoantigen eliciting autoantibodies cross-reactive with HTLV-I gag antigens.

Identification of type-specific linear epitopes in the glycoproteins gp46 and gp21 of human T-cell leukemia viruses type I and type II using synthetic peptides.

Synthetic peptides of 20-25 amino acids were employed in enzyme-linked immunosorbent assays to identify linear epitopes in the external glycoprotein gp46 and the transmembrane glycoprotein gp21 of human T-cell leukemia/lymphotropic viruses type I (HTLV-I) and II (HTLV-II). Ten linear epitopes were identified in the HTLV-I glycoproteins, seven in gp46 and three in gp21. Three major linear epitopes were identified in the gp46 of HTLV-II. Peptides representing linear epitopes of gp46 were found to be sensitive and specific for the detection of antibody and permit serological identification and differentiation of HTLV-I and HTLV-II infections.

Identification of new epitopes recognized by human monoclonal antibodies with neutralizing and antibody-dependent cellular cytotoxicity activities specific for human T cell leukemia virus type 1.

We have generated a number of EBV-transformed B cell lines producing human mAb against human T cell leukemia virus type 1 (HTLV-1) from the peripheral blood B lymphocytes obtained from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. Various synthetic peptides corresponding to antigenic regions of HTLV-1 gag and env proteins were used for the screening of antibodies in ELISA. In our study, four IgG mAb to the gag p19 amino acids 100 to 130, and 5 IgG mAb to the env p46 amino acids 175 to 199 were characterized. An immunofluorescence assay showed that all of these mAb specifically bound to the surface of HTLV-1-bearing cell lines. Among these mAb, one anti-gp46 mAb, designated KE36-11, neutralized the infectivity of HTLV-1 as determined by both the inhibition of HTLV-1-induced syncytium formation and transformation assays in vitro. An antibody-binding assay using overlapping oligopeptides revealed that KE36-11 recognized a new epitope locating between the gp46 amino acid sequence 187-193 (Ala-Pro-Pro-Leu-Leu-Pro-His). Another anti-gp46 mAb, designated KE36-7, showed antibody-dependent cellular cytotoxicity against HTLV-1-bearing cell line. KE36-7 bound strongly to the 10-mer peptide-gp46 187-196, and weakly to peptides containing the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). These two epitopes, which are associated with HTLV-1 neutralization and antibody-dependent cellular cytotoxicity, are thus the first epitopes identified in human HTLV-1 infection. It is possible that passive immunization of humans with these two human mAb are effective on the protection of HTLV-1 infection in vivo.

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-Gag p19(100-130), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, cys-gag p19(100-130), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl bovine serum albumin-cys-gag p19(100-130) conjugate and cys-gag p19(100-130)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was 100-fold more sensitive than the conventional enzyme immunoassay, in which a cys-gag p19(100-130)-bovine serum albumin-coated polystyrene ball was incubated with test serum and, after washing, with (anti-human IgG gamma-chain) Fab'-peroxidase conjugate. The degree of inhibition by preincubation of test sera with excess of cys-gag p19(100-130) in combination with an appropriate cut-off value for the fluorescence intensity of bound beta-D-galactosidase activity discriminated almost all seropositive samples from seronegative ones.

Differential antibody responsiveness to p19 gag results in serological discrimination between human T lymphotropic virus type I and type II.

A new algorithm based upon the differential antibody responses to two gag gene products (p19 and p24) of human T lymphotropic virus (HTLV) has been suggested for serologic discrimination of HTLV type I (HTLV-I) and type II (HTLV-II) [Lillihoj et al., 1990]. To evaluate the practical usefulness of this algorithm, serum specimens from HTLV-seropositive individuals whose infection was confirmed by PCR analysis to be HTLV-I (n = 60) or HTLV-II (n = 61) were analyzed by western blot. The intensities of the antibody response to p24gag and p19gag were scored by one individual without prior knowledge of PCR results. According to the algorithm, specimens with p19 greater than or equal to p24 were classified as HTLV-I, whereas specimens with p19 less than p24 were classified as HTLV-II. Of 60 PCR confirmed HTLV-I specimens, 56 had p19 greater than or equal to p24 (93%) while 4 had p19 less than p24. Of 61 PCR confirmed HTLV-II specimens, 56 had p19 less than p24 (92%) and 5 had p19 greater than or equal to p24. The overall accuracy of serologic differentiation when using this algorithm was 92%, as 4 of 60 HTLV-I (7%) and 5 of 61 HTLV-II (8%) could have been wrongly classified. Although the differential antibody response to p19gag and p24gag provides a simple means of serologically distinguishing between HTLV-I and HTLV-II infection in population-based epidemiological studies, in a clinical context more accurate means of confirmation are required. The dominant p19gag responses were mapped to the C-terminus of p19 (p19(102-117)).(ABSTRACT TRUNCATED AT 250 WORDS)

New approach for generation of neutralizing antibody against human T-cell leukaemia virus type-I (HTLV-I) using phage clones.

We have screened a phage peptide library to address whether clones binding to a monoclonal antibody (mAb) could be isolated and if the selected phage particles would be able to elicit an in vivo immune response against the original antigen. A phage peptide library, consisting of seven random amino acids inserted in the minor coat protein (pIII), was screened for specific binding to a rat mAb LAT-27, which is capable of neutralizing human T-cell leukaemia virus type-I (HTLV-I) by binding to its envelope gp46 epitope, (amino acids LPHSNL). Total 37 clones were selected from the library and one clone named 4-2-22 was tested for its immunogenicity in three rabbits. The all rabbit immune sera showed strong binding activity to a gp46 peptide carrying the neutralization sequence, stained gp46-expressing cells and neutralized HTLV-I in vitro as determined by cell fusion inhibition assay. These results show that the selected phage clone was capable of mimicking the epitope recognized by a HTLV-I neutralizing mAb, and it can be used as an immunogen to induce protective immune response against HTLV-I. Thus, the present methodology could be one of the approaches to develop vaccines against infectious agents in a simple and inexpensive way.