An immunoglobulin G capture assay (GACRIA) for anti-HTLV III/LAV and its use as a confirmatory test

An immunoglobulin G (IgG) antibody capture assay (GACRIA) methodologically distinct from other assays for the detection of antibodies to human T-lymphotropic virus type III/lymphadenopathy-associated virus (anti-HTLV III/LAV) was developed and used to test 1,055 serum samples previously screened by a competitive assay (COMPRIA). Eight hundred and twenty-three (78%) sera were positive and 129 (12.2%) negative in both assays. The coefficient of correlation between the two assays was 0.87, and COMPRIA appeared more sensitive than GACRIA. On retesting 103 sera that gave initially conflicting results, 81 discrepancies were resolved, 77 because of a change in the COMPRIA result. The 22 persistently discrepant samples gave an equivocal or positive result by COMPRIA and were negative by GACRIA. Thirteen of these 22 were positive in a commercial ELISA (ELAVIA). Twenty of them were also tested by Western blot; all were reactive with at least two HTLV III/LAV protein bands. The persistently discrepant samples were of four types: laboratory control sera (n = 2); naturally occurring weakly reactive samples (n = 5); samples that were anti-HTLV III/LAV IgG negative but IgM positive (n = 5; all five individuals from whom these samples were drawn were symptomatic); samples from one laboratory that were probably accidentally contaminated with anti-HTLV III/LAV-positive serum (n = 10). It was concluded that GACRIA for anti-HTLV III/LAV is specific and adequately sensitive and, in conjunction with other assays, is a useful confirmatory test whose format could readily be changed to ELISA.     

HLA-A, B and DR antigen frequencies in patients with AIDS-related persistent generalized lymphadenopathy (PGL) and thrombocytopenia

HLA-A, B, DR phenotype frequencies were studied in 69 homosexual patients with persistent generalized lymphadenopathy (PGL), in 8 patients with LAV/HTLV III related thrombocytopenic purpura and in 21 homosexual controls. Antigen DR5 was significantly increased in PGL patient groups as compared to controls. Our findings suggest that genetic factors associated with HLA-DR5 antigen may predispose individuals infected by the LAV/HTLV III virus to the occurrence of PGL and/or thrombocytopenia.     

HLA typing in patients with chronic adenopathies in a population at risk for AIDS

HLA-A B and DR typing were performed in 77 patients with AIDS related complex (ARC)--69 lymphadenopathy associated syndrome and 8 thrombocytopenic purpura LAV/HTLV III related--and 21 symptom free homosexual males. A significant increase in the frequency of HLA DR5 antigen was observed in patients with ARC mainly in purpura thrombocytopenic patients. We suggest that increase of HLA DR5 antigen support the view that DR5 antigen could be one of the factors necessary at the spreading out clinical symptoms.     

Seroepidemiology of HTLV-III (LAV) in the Federal Republic of Germany

Summary In 1984 10,281 sera were collected in the FRG and examined for antibodies to HTLV-III (LAV) with an enzyme-linked immunosorbent assay and confirmative tests. Of the German AIDS patients 81% have antibodies. Individuals belonging to AIDS risk groups, homosexuals, haemophiliacs and i.v. drug abusers, have antibody frequencies between 25%–72%. The detection of HTLV-III antibodies in blood donours indicates that the virus is being transmitted by blood transfusions.Abbreviations AIDS acquired immunodeficiency syndrome - LAS lymphadenopathy syndrome - ARC AIDS related complex - LAV lymphadenopathy associated virus - HTLV-III human T-lymphotropic virus type III - HBV hepatitis B virus     

Immunological and virological investigation in patients with lymphoadenopathy syndrome and in a population at risk for acquired immunodeficiency syndrome (AIDS), with particular focus on the detection of antibodies to human T-lymphotropic retroviruses (HTLV III)

Thirteen patients affected with unexplained lymphoadenopathy, fever, weight loss, and diarrhea (lymphoadenopathy syndrome; LAS) were evaluated for the possible appearance of acquired immunodeficiency syndrome (AIDS) and for immunological and virological characterization. The patients belonged to categories of individuals at risk for AIDS and were homosexual and/or drug abusers or hemophiliacs. Lymph node biopsy showed the histological picture of a follicular hyperplasia. The study of cell-mediated immunity (CMI), humoral immune response, and natural killer (NK) activity demonstrated a significant decrease in T cells with the helper/inducer phenotype (OKT ₄ ⁺ cells) and a relatively increased number of lymphocytes with the suppressor/cytotoxic phenotype (OKT ₈ ⁺ cells). NK activity was significantly lower than in normal controls. Thein vitro response to policlonal activators (phytohemagglutinin; PHA) and the cutaneous responsiveness to recall skin tests were impaired, whereas immunoglobulin production was increased, mainly in the IgG fraction. Virological studies showed high serum antibody titers to cytomegalovirus (CMV) but a lack of specific CMI as assayed by the leukocyte migration inhibition test (LMIT). CMV was also isolated from the urine specimen of one patient. The antibody pattern to Epstein-Barr virus (EBV) showed the uncommon contemporary presence of both Epstein-Barr nuclear antigen (EBNA) and early antigen (EA) antibodies. Antibodies to human T-lymphotropic retroviruses (HTLV III) were positive in 10 patients and the virus was isolated in 3 of them. In some patients the presence of serum antibodies to HTLV III was not associated with an impairment of the immune function. A group of individuals at risk for AIDS without LAS was also evaluated for the presence of HTLV III antibodies; the percentage of positive sera was 11.4. Nevertheless, individuals without specific antibodies had immunological abnormalities resembling those of LAS HTLV III-positive patients. The possible implications of these findings are discussed.     

Evaluation of a new assay for antibodies to LAV/HTLV III

The sensitivity, specificity and reproducibility of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to LAV/HTLV III produced by Genetic Systems was assessed with the identical panel of sera used in previous evaluations of anti-HTLV III ELISAs. The results from this study show that the Genetic Systems anti-LAV/HTLV III ELISA proved to be of equivalent sensitivity and to have higher specificity than assays currently used in Australia for screening purposes while maintaining high levels of intra- and inter-laboratory reproducibility.     

Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24)

The acquired immunodeficiency syndrome (AIDS) may be transmitted by blood transfusions and by blood products from donors who have been infected with the lymphadenopathy-associated virus (LAV). Such donors may generally be identified on the basis of a positive test for antibodies-against LAV proteins. We have already described an anti-LAV assay based on the use of crude virus-infected tissue culture medium, which avoids elaborate, expensive and potentially hazardous virus purification steps. This test was operationally specific for antibodies to the approximately 24 kD core protein of the virus (P24; Neurath et al. J. Virol. Methods 11, 75, 1985). Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA)--or enzyme-linked immunoassay (ELISA)--inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed.     

Serum interferon and clinical manifestations of infection with human T-lymphotropic virus type III

Sera from 32 homosexual men were studied for the presence of antibodies against humanT-lymphotropic virus type III (HTLV III) and acidlabile interferon (IFN) alpha. Infection with HTLV III was found to be associated with the presence of serum IFN. IFN was detected in 74% of sera from male homosexuals with HTLV III antibody, but in only 6% of sera from antibody-negative individuals. More than 80% of sera from HTLV III-infected patients with the acquired immune deficiency syndrome (AIDS) or pre-AIDS conditions (generalized lymphadenopathy or the AIDS-related complex) were positive for IFN, while IFN was not present in sera from healthy homosexual men with HTLV III antibody. In conclusion, the presence of serum IFN may be predictive of the development of AIDS or pre-AIDS conditions in male homosexuals exposed to HTLV III.     

Effect of beta-propiolactone--an inhibitor of HTLV III/LAV activity--on immunological analyses

beta-Propiolactone (BPL) inactivates LAV/HTLV III, the retrovirus associated with acquired immune deficiency syndrome (AIDS). Addition to specimens from patients with suspected AIDS or antibodies to LAV/HTLV III could reduce any occupational risk to laboratory staff. This study demonstrates that BPL treatment does not significantly affect the immunological analyses commonly required on these patients, namely measurements of serum immunoglobulins, complement components C3 and C4 and other serum proteins, detection of autoantibodies and estimations of T lymphocyte subpopulations.     

Treatment of the acquired immune deficiency syndrome

The acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC) are sequelae of immune system injury initiated by a novel human retrovirus [human T-lymphotrophic virus strain III/lymphadenopathy-associated virus (HTLV III/LAV)]. The resulting spectrum of immune deficiency sets the stage for opportunistic infection and malignancy. In this review, we consider progress made in the treatment and prevention of AIDS and HTLV III/LAV infection. Immunomodulator and antiviral approaches are discussed.     

Performance evaluation of the Abbott HTLV III EIA, a test for antibody to HTLV III in donor blood

An enzyme immunoassay (EIA), designed to detect antibodies to human T-cell lymphotropic virus type III (HTLV III), was evaluated. The antibody test was found to be highly sensitive; serum from 221 of 223 (99.1%) patients with acquired immune deficiency syndrome (AIDS) was positive for antibodies to HTLV III. In addition, the antibody test was found to be highly specific; approximately 99.75% of 20,720 serum or plasma samples from blood donors were negative for antibody to HTLV III. In most cases, the Western Blot analysis agreed well with the EIA. Eighty-one of 82 (98.8%) EIA-positive samples from patients with AIDS were Western Blot positive. Of the EIA-positive blood donors, 21 of 36 (58%) were detected and confirmed by Western Blot analysis. A solid-phase competitive EIA also was evaluated as an alternate procedure. The preliminary results indicate that this immunoassay has a high degree of sensitivity and specificity and could serve as an alternate procedure to detect antibody to HTLV III.     

Enumeration of the T4 positive and T8 positive lymphocytes in AIDS and related syndromes. Comparison of the immunogold and the immunofluorescence techniques

Immunofluorescence and immunogold techniques yielded similar results when used to determine the T lymphocyte subsets (T4 and T8 positive cells) in patients with AIDS or AIDS-related complex and in healthy homosexual men seropositive for HTLV III/LAV antibody. In these pathological situations, the advantages of immunogold staining could render this technique useful in a clinical laboratory.     

HTLV III antibodies and immunological alterations in hemophilia patients

Summary The clinical, immunological, and serological status of 28 patients with hemophilia A and of 13 patients with hemophilia B was investigated. Thirty-four patients were treated regularly by clotting factor concentrates and 7 patients had been substituted only 1 to 4 times. Almost all patients with severe hemophilia suffered from hepatopathy. No patient had clinical evidence of the acquired immunodeficiency syndrom (AIDS).Asymptomatic hemophiliacs showed a decreased number of T-helper (OKT 4) cells and an increased number of T-suppressor (OKT 8) cells, which resulted in an inversed OKT 4/OKT 8 cell ratio. Natural killer cell activity of all patients was decreased compared to controls. After culture there was no significant difference of NK cell activity between hemophiliacs and controls. This phenomen was interpreted as a possible maturation defect of NK-cells in vivo.No relationship between immunological alterations and hepatopathy, hepatitis markers, CMV antibodies, amount and source of required factor concentrates, and the kind of hemophilia was observed. IgG immunoglobulins were higher and the OKT 4/OKT 8 ratio lower in the eight patients with lymphadenopathy than in patients without lymphadenopathy. The prevalence of antibodies to human T-lymphotropic virus (HTLV III) was measured in 35 hemophiliacs and in 25 polytransfused patients, most of whom were suffering from acute leukemia. In 8 of 35 hemophiliacs antibodies to HTLV III virus were detected by an enzyme linked immunosorbent assay (ELISA) and confirmatory tests. All seropositive patients were treated by blood products from the United States. Eight hemophiliacs treated by factor concentrates from German donors only were seronegative. In comparison 2 of 25 examined non-hemophilia patients receiving multiple blood products from local donors were seropositive for HTLV III. The results show that hemophilia patients treated by imported clotting factor concentrates have a high risk of HTLV III positivity. Hemophiliacs substituted by blood products obtained by local donor pools have only a small risk of infection. Because non-hemophiliac polytransfused patients had HTLV III antibodies, there must be asymptomatic virus carriers in the local donor pool. The HTLV III antibody screening of all donors and the heat treating of factor concentrates will give better therapeutic safety.Abbreviations AIDS Acquired immunodeficiency syndrome - ALT Alanin-Aminotransferase - Anti-HBc Antibody to hepatitis B core antigen - Anti-HBs Antibody to hepatitis B surface antigen - AST Aspartat-Aminotransferase - CMV Cytomegaly virus - EBV Epstein-Barr-virus - EDTA Ethylendiamintetraacetate - ELISA Enzyme linked immunosorbent assay - -GT Gamma-Glutamyl-Transferase - HBsAg Hepatitis B surface antigen - HLA Human Leukocyte Antigen - HTLV III Human T-lymphotropic virus - IL-2 Interleukin 2 - IPS Immune peroxidase staining - LDH Lactat-Dehydrogenase - LGL Large granular lymphocyte - LU₃₀ Lytic units - MNC Mononuclear cells - NK Natural Killer cells - OKT 3 Total T-cells - OKT 4 T-helper cells - OKT 8 T-suppressor cells     

Infection with human T lymphotropic virus type I in organ transplant donors and recipients in Spain

Human T-cell lymphotropic virus (HTLV) antibody screening is not recommended uniformly before transplantation in Western countries. In the year 2001, the first cases of HTLV-I infection acquired through organ transplantation from one asymptomatic carrier were reported in Europe. All three organ recipients developed a subacute myelopathy shortly after transplantation. This report rose the question about whether to implement universal anti-HTLV screening of all organ donors or selective screening of donors from endemic areas for HTLV-I infection should be carried out. A national survey was conducted thereafter in which anti-HTLV antibodies were tested in 1,298 organ transplant donors and 493 potential recipients. None was seropositive for HTLV-I and only one recipient, a former intravenous (i.v.) drug user, was found to be infected with HTLV-II. In a different survey, HTLV screening was conducted in 1,079 immigrants and 5 (0.5%) were found to be asymptomatic HTLV-I carriers. All came from endemic areas for HTLV-I infection. No cases of HTLV-II infection were found among immigrants. These results support the current policy of mandatory testing of anti-HTLV antibodies in Spain only among organ transplant donors coming from HTLV-I endemic areas or with a highly suspicion of HTLV-I infection.     

Immunological status of hemophiliacs: study of blood T-lymphocyte populations, serum immunoglobulins, and the prevalence of anti-LAV antibodies

Blood T-lymphocyte subsets and serum immunoglobulin levels were studied in a group of 52 haemophiliacs (44 patients with haemophilia A and 8 patients with haemophilia B). None of the patients had AIDS or belonged to any AIDS high-risk group. Patients were exclusively treated with clotting fractions obtained from healthy volunteers in metropolitan France. As compared to a group of 52 normal donors, haemophiliacs had increased numbers of suppressor lymphocytes, which resulted in depressed helper/suppressor (H/S) ratios, and increased levels of serum IgG and IgA. 21 haemophiliacs (40,3%) had a H/S ratio less than 1.4. Among patients with haemophilia A a higher mean IgG level was found in patients presenting lymphadenopathy. Decreased mean H/S ratio and increased mean serum IgG level were found in patients receiving more than 50 000 U of factor VIII during the 2 years preceding the study. No striking difference in mean serum IgG, IgA and IgM levels was found in patients with haemophilia A when H/S ratios were higher and lower than 1.4 respectively. As AIDS and immunological abnormalities among haemophiliacs probably share a common viral origin, this study emphasize the need to discourage blood donation from donors who belong to any AIDS high-risk group, and to screen sera from the blood donor population for antibodies to LAV/HTLV III.     

Early Defect of Phagocytic Cell Function in Subjects at Risk for Acquired Immunodeficiency Syndrome

We studied the functions of peripheral blood monocytes and polymorphonuclear cells in 15 apparently healthy homosexual men, eight homosexual or bisexual subjects with unexplained generalized lymphadenopathies (pre-AIDS), four homosexual men with acquired immunodeficiency syndrome (AIDS), and 15 heterosexual men. In comparison with normal controls, the homosexual groups studied presented a decreased monocyte candidacidal activity for Candida pseudotropicalis that gradually deteriorates as the clinical symptoms progress towards AIDS. The monocyte phagocytic function was retained. Although the phagocytic and candidacidal activities of the polymorphonuclear cells did not differ from those of the normal controls, the candidacidal activity in some of the cases studied was unusually enhanced, indicating that the cells were in an activated state. In addition, only two of nine sera tested from asymptomatic homosexual males were positive for antibodies to HTLV-III/LAV, while six out of eight pre-AIDS and both of the two AIDS patients tested had antibodies to AIDS-associated retrovirus. We suggest that in AIDS the phagocytic system is already involved, together with B and T lymphocyte abnormalities, during the early events of the syndrome, even without the detection of AIDS-associated retrovirus antibodies.     

Prevalence of HIV antibodies in groups at risk in Zürich,Switzerland

Summary The prevalence of HIV antibodies in various groups at risk was studied in 1,546 persons in Zürich. The prevalence was 17% (39/236) in homosexual men, 7% (13/180) in bisexual men, and 45% (14/31) and 42% (22/53) in female and male intranvenous drug abusers, respectively. Heterosexual transmission appeared to be the route of infection in four seropositive persons (two women and two men) who had no homosexual contacts and were not drug abusers (4/1050).Abbreviations HIV Human immune deficiency virus - HTLV III Human T-cell lymphotropic virus type III - IVDA Intravenous drug abusers - LAV Lymphadenopathy-associated virus     

Glycosylation pattern of herpes simplex virus type 2 glycoprotein G from precursor species to the mature form

Summary Studies of adult T-cell leukaemia virus/human T-cell leukaemia/lymphotropic viruses (ATLV/HTLV-I) in Japan indicate that the virus is involved only with the development of ATL. By contrast, reports from the U.S.A. about HTLV have from time to time claimed that related HTLV are concerned not only with ATL of black persons, but also with a wide range of diseases, such as mycosis fungoides/Sezary's syndrome, T-cell hairy cell leukaemia, acquired immune deficiency syndrome (AIDS) and also multiple sclerosis. Using morphological, biological, serological and molecular hybridisation studies, we were able to confirm that the viruses implicated in the development of ATL and AIDS are distinct and that ATLV/HTLV-I is involved only in ATL, and HIV/LAV/HTLV-III only in AIDS. In vitro, ATLV/HTLV-I transformed and immortalised T-cells, while HIV/LAV/HTLV-III killed our T-cells. Failure to detect any serological cross-reaction indicates that all the structural proteins are different. Likewise, Southern blot studies failed to reveal any cross-hybridisation. Sixty patients with multiple sclerosis failed to reveal any association with ATLV/HTLV-I or with HIV/LAV/HTLV-III. Our conclusion is that ATLV/HTLV-I is involved only in ATL of Japanese and of some black persons of African origin, and that HIV/LAV/HTLV-III is associated only in AIDS.     

Primary isolation of human T-cell leukemia-lymphoma virus types I and II: use for confirming infection in seroindeterminate blood donors.

We describe the use of an immunofluorescence assay and coculture to confirm human T-cell leukemia-lymphoma virus (HTLV) infection. Peripheral blood mononuclear cells from 32 of 32 seropositive donors were positive in the immunofluorescence assay, and 63% of their cocultures produced p24 antigen. Specific antibodies distinguished HTLV type I (HTLV-I) from HTLV-II. HTLV-I or HTLV-II was isolated from donors with indeterminate serologic test results.     

Sensitivity and specificity of commercial ELISA kits for screening anti-LAV/HTLV III

Two panels consisting of 236 and 258 anti-LAV/HTLV III positive and 64 and 50 negative sera, respectively (determined in Western blots), were tested with 8 different, commercially available ELISA test kits for detecting LAV/HTLV III antibodies (Abbott, Dupont, Electro-Nucleonics, Litton, Organon, Pasteur, Sorin, Wellcome). Positive sera were selected for levels of antibodies ranging from average to very high, and the negative sera included both negative sera and sera with antibodies to cellular components such as HLA antigens or nuclear membranes. In examination of the 2 panels, the sensitivity of the tests ranged from 97.2 to 100%, and the specificity from 70.0 to 100%. No test was ideal.