Clonal selection of human immunodeficiency virus (HIV): serological differences in the envelope antigens of the cloned viruses and HIV prototypes (HTLV-III B, LAV, and ARV)

Human T-cell lymphotropic virus type III B (HTLV-III B) derived from H9/HTLV-III cells was cloned by a plaque-forming assay. Cloned viruses showed different restriction cleavage patterns, suggesting that HTLV-III B is a mixture of several viruses. Using two different neutralization assay systems, we compared the envelope antigenicity of uncloned HTLV-III B, cloned viruses, lymphadenopathy-associated virus (LAV), and acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV). A diversity of viral envelope antigens was consistently found among the strains of these AIDS-related retroviruses. These results concur with the previous finding that genomic divergence of AIDS-related viruses is located mainly in the region of the viral envelope gene. Thus, this plaque assay is useful for the clonal analysis of the virus, which is essential for comparing the envelope antigen(s) of the virus.     

Nonrandom association of cellular antigens with HTLV-III virions

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.     

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     

Tumor promoter, TPA, enhances replication of HTLV-III/LAV

The incubation of Molt-4/HTLV-III cells, human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV)-producer cell line, with more than 0.5 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 to 4 days stimulated virus-induced cell killing which resulted in a high production of HTLV-III/LAV. TPA significantly increased the number of plaque-forming viruses released from the cultures as well as viral RNA content. Interestingly, MT-4 cells freshly infected with HTLV-III/LAV treated for 4 days with 0.125 to 2.0 ng/ml of TPA were found to retain the capacity to grow, because TPA inhibited the induction of the virus-specific antigens and cytopathic effects. In contrast to the situation in infected MT-4 cells in liquid cultures, the addition of 0.125 and 0.25 ng/ml of TPA into agarose medium induced large plaques, suggesting that TPA basically enhanced the production of the virus from one infected MT-4 cell. Taken together, the data suggest that TPA enhances the replication of HTLV/III/LAV. These assay systems are shown to be useful for analyzing the induction or suppression of a virus infection by many drugs and other factors in vitro.     

The Walter Reed staging classification for HTLV-III/LAV infection

Patients with acquired immunodeficiency syndrome (AIDS) represent a minority of those who have been infected with human T-lymphotropic virus type III (HTLV-III). The clinical presentation of patients with HTLV-III infections can range from asymptomatic through chronic generalized lymphadenopathy, to subclinical and clinical T-cell deficiency. The US Army has recently adopted a staging classification for HTLV-III infection. The purpose of the Walter Reed (WR) staging Classification is to provide uniformity for routine clinical evaluation of military personnel with HTLV-III infection, to facilitate understanding of the natural history of these infections, and to help evaluate the clinical response to antiviral treatment regimens. Stage WR0 designates high-risk contacts, while stages WR1-6 require documentation of HTLV-III infection. Stages WR1-6 show ascending degrees of disease, so that those classified in WR6 manifest antibodies to HTLV-III, chronic lymphadenopathy, T helper cell counts below the normal limit, delayed hypersensitivity, thrush, and opportunistic infection. HTLV-III infections with symptoms are designated by the addition of the letter B, while the occurrence of Kaposi's sarcoma is designated by adding the letter K. This classification scheme is based on the fact that the T helper cell is the principal target cell of HTLV-III and the clinical observation that the functional integrity of the T helper cell determines the clinical presentation. Preliminary studies in 39 sequential patients followed for over 18 months found that patients who entered in stages WR3-WR6 had a slow but progressive course to the next stage or death. Many questions about clinical progression remain, but it is hoped that this staging classification will facilitate their resolution.     

HTLV-III/LAV Viral Antigens in Lymph Nodes of Homosexual Men With Persistent Generalized Lymphadenopathy and AIDS

The presence of core antigens of retrovirus HTLV-III/LAV, referred to as "AIDS-related virus" (AV), has been sought in lymph node samples of patients with persistent generalized lymphadenopathy (PGL, 28 patients), prodromal AIDS (1 patient) and AIDS with Kaposi sarcoma (3 patients). In 30 patients the deposition of viral antigens, detected by monoclonal antibodies to HTLV-III and LAV, could be observed within the germinal centers (GCs) primarily within the extracellular network of immune complexes, and the two patients who were negative were atypical. No AV could be found in normal tonsil or in samples with follicular hyperplasia of unknown etiology (20 cases). These findings, taken together with the ultrastructural identification of typical retrovirus particles in all 9 PGL and 2 AIDS cases studied, indicates that the network of follicular dendritic (FD) cells is an important reservoir of AV virus antigen at this site. The persistence of this retrovirus inside the GCs helps explain how the follicular hyperplasia affecting FD cells and B blasts in PGL may in progressive cases be accompanied by destruction of FD cells and gradual development of T4+ lymphopenia. T4+ T cells may circulate through the GCs and become infected with AV there. In addition, the identification of retrovirus antigen in situ may be of diagnostic value.     

Time relationship of immune changes to HTLV-III/LAV seroconversion in patients with hemophilia

Longitudinal immune studies of patients with hemophilia A were begun in 1982 by the Regional Hemophilia Center in St. Louis, Missouri. Serum samples collected from 74 participants between 1982 and 1985 were analyzed for antibody to human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV). The incidence of antibody to HTLV-III/LAV has increased significantly in this population of patients with hemophilia. Only one of eight hemophiliacs had detectable antibody before July 1982, whereas 88.7% (55/62) were positive in 1985. T-cell surface markers were markedly abnormal in seropositive hemophilia patients with decreased percentage and number of OKT4-positive cells compared with seronegative hemophiliacs and controls. Lymphoproliferative responses to mitogens and antigens were normal in seronegative hemophilia patients. Seropositive hemophiliacs, compared with seronegative hemophiliacs, had significantly decreased lymphoproliferative responses, especially to pokeweed mitogen, tetanus, and Candida stimulations. Immune studies of seven HTLV-III/LAV seropositive hemophiliacs revealed antigen unresponsiveness and decreased T4 cells 2 to 32 months prior to development of full-blown AIDS. Longitudinal immune studies from 1983-85 revealed increasing number of seropositive hemophiliacs with antigen unresponsiveness and decreased T4 cells.     

Efficient replication of HTLV-III and STLV-IIImac in human Jurkat cells

High producer cell lines were established by infecting Jurkat cells with either HTLV-IIIB from H9 cells or with STLV-IIImac from HUT-78 cells. The Jurkat cells produced both viruses at least 10 times more efficiently then the original cell lines; the pelleted virus from Jurkat cells was also less contaminated with non-virion proteins. Accordingly, a higher specificity was attained in an ELISA for antibodies to HTLV-III if the antigenic activity was derived from the virus from Jarkat cells, as opposed to that from H9 cells.     

Sensitive assay for neutralizing antibodies against AIDS-related viruses (HTLV-III/LAV)

A sensitive assay for neutralizing antibodies (NA) against AIDS-related viruses (HTLV-III and LAV) was developed, using human T-cell lymphotropic virus type-I (HTLV-I)-bearing and HTLV-III-susceptible MT-4 cells. NA to HTLV-III in 21 patients with acquired immune deficiency syndrome (AIDS), 10 individuals with AIDS-related complex (ARC), 20 healthy male homosexuals, and 10 healthy male controls were titrated. Antibodies to HTLV-III were also detected by indirect immunofluorescence (IF). The assay was sensitive up to a dilution of 1:10 000. Sera from patients with AIDS showed a geometric mean titer (GMT) of NA of 1:475, whereas much higher GMTs (1:1318 and 1:1009) were observed in patients with ARC and healthy male homosexuals, respectively. Moreover, titers of NA significantly correlated with the levels of anti-HTLV-III antibodies detected by IF.     

The LAV/HTLV-III virus may evade elimination by the immune system by inducing low zone tolerance to itself

The acquired immunodeficiency syndrome (AIDS) is caused by the LAV/HTLV-III virus. The incubation period for AIDS is prolonged and on the order of years. We hypothesize that during this prolonged incubation period the LAV/HTLV-III virus is replicating very slowly and is present in extremely low concentrations. The concentrations of the virus may be low enough that the virus induces a low zone tolerance to itself in the T-cell arm of the immune system. B-cells which are resistant to direct low dose tolerance induction may remain responsive to the LAV/HTLV-III virus in a direct fashion without specific helper T-cells. Thus, anti-LAV/HTLV-III antibody may be produced even though the more important cellular immune response has been crippled by the virus. We also outline two hypothetical approaches for breaking this tolerance and restoring the cellular immune response to the LAV/HTLV-III virus.     

Pathologic features of AIDS encephalopathy in children: evidence for LAV/HTLV-III infection of brain

The neuropathologic findings in 11 children with a new CNS disorder that occurs in children with the acquired immunodeficiency syndrome (AIDS) and is postulated to be due to LAV/HTLV-III, the virus that causes AIDS, are reported. The children, who ranged in age from 4 months to 11 years, died of AIDS complicated by progressive encephalopathy. Ten of the children either had positive serum antibody for LAV/HTLV-III or had received blood products from donors later found to be antibody-positive. Examination of the brains of these children at autopsy revealed a unique constellation of findings, including varying degrees of diminished brain weight in all cases, inflammatory cell infiltrates in nine brains, multinucleated cells in eight, three of which also contained multinucleated giant cells, vascular calcification in ten, vascular and perivascular inflammation in five, and white matter changes in nine. Inflammatory and vascular lesions were most prominent in basal ganglia and pons. LAV/HTLV-III retroviral particles, associated with multinucleated giant cells, were observed in two brains on electron microscopic examination. These two and one additional brain had evidence of the LAV/HTLV-III genome by hybridization studies. Only one brain had a recognizable opportunistic infection.     

Detection of HTLV-III/LAV antigens in peripheral blood lymphocytes from patients with AIDS

Summary HTLV-III was searched for in frozen sections of peripheral blood lymphocytes obtained from AIDS patients by an immunofluorescence technique. Human IgG against HTLV-III/LAV and monoclonal antibodies against HTLV-III/LAV P24 antigen, yielded a strong cytoplasmic fluorescence in frozen sections of the lymphocytes. Some cells containing HTLV-III antigens displayed multinucleated giant forms. They also reacted with monoclonal antibodies against helper/inducer T-cells (OKT 4⁺), as demonstrated by direct double staining immunofluorescence. Similarly, complexes of immunoglobulins and C₃ component of complement were also detected on HTLV-III/LAV Ag expressing lymphocytes.Immunofluorescence study of frozen sections of peripheral blood lymphocytes appeared to be a simple, fast and reliable method for detection of HTLV-III/LAV Ag in AIDS patients.With 3 Figures     

Lymphadenopathy and antibodies to HTLV-III in homosexual men

Summary The study provides information on the epidemiology of HTLV-III infection and the lymphadenopathy syndrome (LAP) in 374 German homosexual men. Sexual contacts in the USA and rectal enemas before receptive anal intercourse are the main risk factors associated with virus transmission. HTLV-III seropositivity is significantly correlated with LAP. Prominent clinical signs are infreqquent. Immunological and haematological abnormalities are prevalent, and the retrovirus infection is frequently associated with serological markers of other viruses (hepatitis B, herpes group viruses). Lymphadenopathy as a manifestation of HTLV-III infection is discussed within the context of AIDS-related disorders.Abbreviations AIDS Acquired Immunodeficiency Syndrome - CMV Cytomegalovirus - EBV Epstein-Barr virus - HBV Hepatitis B virus - HTLV-III Human leukaemia retrovirus type III - LAP Lymphadenopathy     

Stages in LAV/HTLV-III Lymphadenitis

The aim of the present study was to investigate the relation between the histopathological findings in LAV/HTLV-III lymphadenitis and immunological, clinical, and serological variables. The study group included 38 consecutive homosexual men with persistent generalized lymphadenopathy (PGL) in whom lymph node biopsy was performed. The histopathological lymph node changes were grouped into three stages. Opportunistic infections at the time of biopsy and their development during follow-up were significantly associated with stage III histology (follicular depletion). Analysis of blood from 10 patients with stage III histology revealed significantly (P less than 0.01) decreased proliferative responses of lymphocytes to mitogens and reduced absolute number of CD5+ and CD4+ lymphocytes compared with 17 patients with stage I histology (follicular hyperplasia), whereas patients with stage II histology (follicular involution) had intermediate values. The absolute number of CD8+ lymphocytes was increased in all three stages, as was IgG, while increase in IgM and IgA was restricted to stage III. No difference was observed between the different histopathological stages with respect to the specificity of the anti-LAV/HTLV-III antibody as measured by immunoblotting. In conclusion, the defects of lymphocytes from the blood of LAV/HTLV-III infected persons reflect alterations in secondary lymphoid tissue. Further, there is a close correlation between these alterations and the clinical status of the patients.     

Stages in LAV/HTLV-III Lymphadenitis

Lymph nodes from 40 homosexuals with persistent, generalized lymphadenopathy were studied for histological and immunohistological changes and classified into histological stages based on the progressive destruction of lymph node follicles in association with progression of the disease. Three patterns were recognized: stage I was characterized by follicular hyperplasia, mantle zone depletion, and follicular fragmentation in the absence of vasculitis, stage II by signs of follicular involution, and stage III by depletion of follicles and dendritic reticulum cells with development of diffuse pattern. The T zone was gradually depleted of CD4-positive lymphocytes, but on further progression, lymphocytic depletion (which also involved the CD8-positive cells) and fibrosis prevailed. The 40 lymph nodes from homosexuals were classified as stage I in 18 cases, stage II in 11, and stage III in 10. One case did not fulfil our histological criteria for LAV/HTLV-III lymphadenitis, although this patient was seropositive. Convincing correlation was found between histological stages and clinical and laboratory data. The triad of follicular hyperplasia, mantle zone depletion, and follicular fragmentation, in the absence of vasculitis, appears pathognomonic for the early disease. The diffuse pattern, however, may be seen in different disease entities.     

Lack of transmission of HTLV-III/LAV infection to household contacts of patients with AIDS or AIDS-related complex with oral candidiasis

To determine the risk of transmission of human T-cell lymphotropic virus Type III/lymphadenopathy-associated virus (HTLV-III/LAV) to close but nonsexual contacts of patients with the acquired immunodeficiency syndrome (AIDS), we studied the nonsexual household contacts of patients with AIDS or the AIDS-related complex with oral candidiasis. Detailed interviews, physical examinations, and tests for serum antibody to HTLV-III/LAV were performed on 101 household contacts of 39 AIDS patients (68 children and 33 adults), all of whom had lived in the same household with an index patient for at least three months. These contacts had shared household items and facilities and had close personal interaction with the patient for a median of 22 months (range, 3 to 48) during the period of presumed infectivity. Only 1 of 101 household contacts--a five-year-old child--had evidence of infection with the virus, which had probably been acquired perinatally rather than through horizontal transmission. This study indicates that household contacts who are not sexual partners of, or born to, patients with AIDS are at minimal or no risk of infection with HTLV-III/LAV.     

Interleukin 2 production in HTLV-III/LAV infection: evidence of defective antigen-induced, but normal mitogen-induced IL-2 production.

We studied mitogen- and antigen-induced interleukin 2 (IL-2) production in HTLV-III/LAV infected and non-infected individuals and compared the results with T cell subpopulations, and with mitogen- and antigen-induced DNA synthesis and production of leucocyte migration inhibitory factor (LIF) in order to understand the controversial findings related to IL-2 production in HTLV-III/LAV infection. The HTLV-III/LAV antibody positive group showed immunological defects: low T helper (Th) cells, high T-suppressor (Ts) cells, reduced mitogen- and antigen-induced DNA-synthesis, but LIF production comparable to the HTLV-III/LAV antibody negative group. The total amount of IL-2, produced either as a response to a mitogenic stimulus or as a response to a soluble antigenic (purified protein derivative of tuberculin, PPD) stimulus, was lower in the HTLV-III/LAV antibody positive group. However, adjusting the IL-2 production to the amount of Th-cells showed that the IL-2 produced by a standard number of Th-cells after mitogen induction was similar in HTLV-III/LAV infected and non-infected individuals. In contrast, the ability of Th-cells of infected persons to produce IL-2, or to proliferate as a response to a soluble antigenic stimulus, was considerably diminished. We conclude that HTLV-III infection leads to a selective incapability to mount a specific Th-cell response either due to an intrinsic defect in the Th-cell population or due to metabolic and/or functional disturbances in antigen-processing and presenting accessory cells.     

Prevalence of HTLV-III/LAV antibodies in Italian asymptomatic hemophiliacs given commercial concentrates of factors VIII and IX

The prevalence of HTLV-III/LAV antibodies was evaluated in 222 Italian asymptomatic hemophiliacs treated exclusively with commercial factor concentrates made with American plasma. An increase of HTLV-III/LAV seropositivity from 1983 to 1985 was evident. This was independent of the type of hemophilia (A or B) but directly correlated to the amount of concentrate administered in the previous year. Immunological data (T4 and T8 lymphocyte subsets) were evaluated in relation to seropositivity to HTLV-III/LAV. The presence of antibodies was found to be significantly associated with a low T4/T8 ratio and to a reduced T4 subpopulation, whereas increased levels of T8 lymphocytes correlated weakly with seropositivity. Data from 47 hemophiliacs tested both in 1984 and 1985 showed that 100% of those negative in 1984 and highly transfused in the previous year seroconverted up to 1985, whereas 33% of those not highly transfused did so.     

Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications

Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus-producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV-III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes.