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.

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.

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.

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.

Prevalence of antibodies to hepatitis C virus, HIV and human T-cell leukaemia/lymphoma viruses in injecting drug users in Tayside, Scotland, 1993-7.

The prevalence of blood-borne viruses in injecting drug users (IDUs) in Tayside, Scotland was determined by testing serum samples from IDUs who underwent attributable HIV antibody testing during 1993-7. The prevalence of antibodies to HIV was 29/802, (3.6%); to hepatitis C virus (HCV) 451/691, (65.3%); and to human T-cell leukaemia/lymphoma viruses type 1 and 2 (HTLV) 0/679, (0.0%). The prevalence of HIV and HCV antibodies were higher in subjects over the age of 25 (P = 0.03 and P = 0.001, respectively). During 1993-7 the prevalence of HCV fell only in younger female IDUs (P < 0.01). HIV prevalence has declined dramatically since 1985, when a rate of 40% was recorded in similar populations. Harm reduction measures have failed to control HCV the spread of infection among IDUs in Tayside, as indicated by the high proportion of antibody positive IDUs, particularly males under the age of 25. Future studies should address the nature and effective reduction of continuing risk taking among IDUs in Tayside.

Viral markers and use of factor products among Finnish patients with bleeding disorders.

In the seventh national voluntary cross-sectional survey (in 1999) of Finnish patients with haemophilia A or B, type 3 von Willebrand disease or factor XIII deficiency, a plasma sample was received from 193 patients (67%). The samples were tested for hepatitis B and C, human immunodeficiency virus (HIV) and human T-cell leukaemia virus (HTLV) antibodies. Fifty-one percent of the patients were hepatitis C antibody positive and 34% hepatitis B core antibody positive. None of the patients had antibodies against HIV or HTLV. Eighteen percent of the patients had an elevated alanine aminotransferase activity. Abnormal alanine aminotransferase was significantly associated with hepatitis C seropositivity. No new seroconversions were detected among the haemophiliacs or patients with type 3 von Willebrand disease when compared with the last two surveys in 1993 and 1996, and there was no seroconversion in sole users of solvent/detergent-treated factor products. Currently, 32% of the patients use prophylactic factor treatment as their principal mode of therapy, particularly the younger patients with severe forms of the bleeding diseases.

The HTLV-I orfI protein is recognized by serum antibodies from naturally infected humans and experimentally infected rabbits.

The mechanism of T-cell transformation by human T-cell lymphotropic virus type I (HTLV-I), though not completely understood, appears to involve the interactions of several viral and cellular proteins. One of these viral proteins, p12(I), encoded by HTLV-I orfI, is a weak oncogene that binds the 16-kDa subunit of the vacuolar ATPase and interacts with the immature beta and gamma(c) chains of the IL-2 receptor. We have expressed the singly spliced orfI cDNA in the baculovirus system and used the recombinant protein as a tool to assess the presence of antibodies in naturally or experimentally infected hosts. In addition, rabbit antisera were raised against various p12(I) synthetic peptides and used to identify three antigenic regions within p12(I), one between the two putative transmembrane regions of p12(I) and two at the carboxy-terminus of the protein. More importantly, sera from a naturally infected human (1 of 32) and experimentally infected rabbits (9 of 20) recognized the rp12(I), demonstrating orfI expression and immunogenicity in vivo. Taken together these data provide the first evidence of orfI expression during HTLV-I infections.

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.

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.

The humoral immune response to human T-cell lymphotropic virus type 1 envelope glycoprotein gp46 is directed primarily against conformational epitopes.

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.

Transmission of human T-cell leukemia virus type 1 to mice.

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10(5) spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.

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.

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.

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 rountine 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 percent, 95 percent confidence limits 0.083 percent to 0.30 percent) and HTLV-2 antibodies in 2 (0.03 percent) sera. Specimens from 8 to 821 (0.97 percent, 95 percent confidence limits 0.42 percent to 1.9 percent). Afro-Caribbean women, three of 1,136 (0.26 percent, 95 percent confidence limits 0.055 percent to 0.78 percent). African women, and one of 3,049 (0.033 percent, 95 percent confidence limits 0.006 percent to 0.18 percent). 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.(AU)

Differential serological diagnosis of HTLV-I and HTLV-II infection by external membrane protein peptide-based enzyme immunoassays.

BACKGROUND: HTLV antibody screening assays detect both antibodies to the etiological agent of adult T-cell leukemia and tropical spastic paraparesis HTLV-I and to the less pathogenic HTLV-II. It is critical to make a differential diagnosis of the two viruses. OBJECTIVES: To design and evaluate synthetic core and envelope-derived peptide enzyme immunoassays (EIA) for serological differential diagnosis. STUDY DESIGN: Peptide EIAs were evaluated with a panel of 202 plasma samples comprised of HTLV antibody positive, serologically classified as confirmed, indeterminate, or non confirmed, characterized as HTLV-I, HTLV-II or neither by genomic amplification. The peptide EIA with the best performance was further used to differentiate between HTLV-I and HTLV-II antibodies in 807 samples from 18 countries in four continents and to provide ratios between the two infections. RESULTS: The gp46 peptide EIA correctly identified 96.5% of HTLV-I and 98.6% of HTLV-II antibody-confirmed samples. HTLV-I was found exclusively in Japan and Caribbean countries; almost exclusively in Africa. HTLV-II represented 10-25% of samples from Canada, Chile and Venezuela and was predominant in the US. CONCLUSIONS: Differential diagnosis between HTLV-I and HTLV-II can be reliably performed using specific peptides from the gp46 envelope protein of each virus.

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region.

Rabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80-103 but not in those to HTLV-II Env gp46, 171-196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80-103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1 x 10(7) cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.

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.

Dependency of antibody titer on provirus load in human T lymphotropic virus type I carriers: an interpretation for the minor population of seronegative carriers.

To evaluate the prevalence of seronegative carriers of human T lymphotropic virus type I (HTLV-I), buffy coat samples from 1015 Okinawan high school students were tested by immunoassays and nested polymerase chain reaction (PCR). Among 17 HTLV-I carriers, 1 person who was seronegative and 1 who was PCR-negative were identified. gag and tax/rex PCR titers correlated with each other (r = .92; P < .001). Of the 17 carriers, 14 (82%) had high virus loads (geometric averages, 522 gag and 703 tax/rex copies/micrograms of DNA; 95% confidence intervals, 38-7260 and 75-6594, respectively). Carriers with low virus loads had < or = 2.2 gag copies. In the high-virus-load group, the gag PCR titers correlated with the antibody titers (r = 0.88; P < .001). The regression line intersected the minimum antibody detection level at 35 gag copies/micrograms of DNA. These results suggest that a small percentage of carriers may be seronegative.