Sera from HTLV-III/LAV antibody-positive individuals mediate antibody-dependent cellular cytotoxicity against HTLV-III/LAV-infected T cells

The causative agent of the acquired immunodeficiency syndrome (AIDS) has been shown to be a human retrovirus called human T lymphotropic virus (HTLV)-III or lymphadenopathy-associated virus (LAV). The nature of the protective immune response against this virus is currently unknown. We report here results using an antibody-dependent cellular cytotoxicity (ADCC) assay which has been developed for measuring a specific immune response against HTLV-III/LAV. Forty-four sera were examined for their ability to mediate ADCC against HTLV-III/LAV-infected T cells. Sera from healthy HTLV-III/LAV seropositive individuals in the presence of mononuclear cells from healthy HTLV-III/LAV seronegative donors exhibited significantly higher levels of ADCC activity compared to sera from patients with AIDS. Western blot analysis of serum samples indicated that antibody reactivity with the p24 protein of HTLV-III/LAV correlated with higher levels of ADCC activity than did reactivity with Gp120/160. The observation that sera from healthy HTLV-III/LAV seropositive individuals mediated higher levels of ADCC activity than did sera obtained from subjects with AIDS suggests that ADCC may represent a protective immune response to infection with HTLV-III/LAV.     

Expression of the art gene protein of human T-lymphotropic virus type III (HTLV-III/LAV) in bacteria

Human T-lymphotropic virus type III (HTLV-III/LAV or HIV) contains a gene designated art (anti-repressor transactivator). Here, we report the expression of the art gene product in bacteria and show that the 20-kilodalton (kDa) bacterially expressed art protein is recognized by serum of a patient. The bacterially synthesized art protein competed in an immunological reaction with a 20-kDa protein produced in HTLV-III/LAV-infected lymphocytes. Antiserum to a synthetic oligopeptide corresponding to a sequence in the second exon of the art gene also precipitated the 20-kDa protein in HTLV-III/LAV-infected cells. These results demonstrate that the 20-kDa art gene product is expressed in cell lines that produce HTLV-III/LAV virions.     

Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV-III/LAV and other lentiviruses.

A retrovirus has been isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma in 1982 at the University of Washington primate center. This isolate, designated MnIV (WPRC-1) (M. nemestrina immunodeficiency virus, Washington Primate Research Center) shows the characteristic morphology of a lentivirus and replicates to high titers in various lymphocyte lines of human and primate origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MnIV revealed multiple bands of structural proteins, including a major viral gag protein of 28 kilodaltons, that did not comigrate with the viral proteins of a human immunodeficiency virus (HIV [FRE-1]) that was also isolated on HuT 78 cells. The relatedness of MnIV to other lentiviruses (HTLV-III/LAV, EIAV, and visna) was examined in radioimmunoassays, by immunoblot techniques, and by N-terminal amino acid sequence analysis of the viral p28 gag protein. The immunoassays revealed cross-reactivity only between MnIV p28 and HTLV-III/LAV p24, and sequence analysis showed that 14 of the 24 N-terminal residues of MnIV p28 and HTLV-III/LAV p24 are identical. These results indicate that MnIV belongs to the same lentivirus family as HTLV-III/LAV but is only partially related to these human acquired immune deficiency syndrome retroviruses.     

Molecular evolution and phylogeny of the human AIDS viruses LAV,HTLV-III,and ARV

Summary A phylogenetic tree for the human lymphadenopathy-associated virus (LAV), the human T-cell lymphotrophic virus type III (HTLV-III), and the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) has been constructed from comparisons of the amino acid sequences of their gag proteins. A method is proposed for estimating the divergence times among these AIDS viruses and the rates of nucleotide substitution for their RNA genomes. The analysis indicates that the LAV and HTLV-III strains diverged from one another after 1977 and that their common ancestor diverged from the ARV virus no more than 10 years earlier. Hence, the evolutionary diversity among strains of the AIDS viruses apparently has been generated within the last 20 years. It is estimated that the genome of the AIDS virus has a nucleotide substitution rate on the order of 10^–3 per site per year, with the rate in the second half of the genome being double that in the first half.     

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.     

Neurology of AIDS virus infection: a clinical classification

Infection with the AIDS virus itself (HIV, HTLV-III, LAV, ARV) is associated with a full spectrum of neurological disorders. The application of diagnostic studies for HTLV-III infection has demonstrated that these neurologic disorders can be the first manifestation of AIDS or occur in the absence of AIDS. The most common conditions associated with HTLV-III infection alone are a subacute encephalopathy (AIDS dementia) and peripheral neuropathy; however, vacuolar myelopathy and both acute and chronic aseptic meningitis are also common. Congenital (or neonatal) transmission of the virus can result in a mental retardation syndrome of delayed onset. The AIDS virus is neurotropic as well as targeting T-helper lymphocytes. The virus has been readily identified in neural tissues and cerebrospinal fluid, including instances in which other central nervous system infections, such as toxoplasmosis, coexist. Hence, recognition of an appropriate syndrome, neurodiagnostic studies, and exclusion (or treatment) of other infections, as well as evidence for HTLV-III infection are required for diagnosis. The development of successful therapy will require agents which cross the blood-brain barrier.     

Serological differences between human T-lymphotropic virus type-I (HTLV-I) and HTLV-III/LAV

Sera from each of five preselected groups of patients with acquired immune deficiency syndrome (AIDS), AIDS-related complex (ARC), hemophilia, adult T-cell leukemia (ATL), and healthy controls were examined for antibodies to human T-cell leukemia (T-lymphotropic) virus type-I (HTLV-I) and HTLV-III by indirect immunofluorescence (IF) and radioimmunoprecipitation (RIP) methods. All sera from five patients with AIDS, ARC, and hemophilia reacted at titers from 1 : 512 to 1 : 5,120 with fixed H9/HTLV-III cells by IF but not with fixed MT-1 cells carrying HTLV-I. Similarly, sera from patients with AIDS, ARC, and hemophilia precipitated HTLV-III-specific polypeptides of 120K, 46K, and 24K. In contrast, sera from five patients with ATL did not react with fixed H9/HTLV-III cells, but reacted with fixed MT-1 cells. Moreover, HTLV-I-specific polypeptides of 68K, 28K, and 24K were precipitated with sera from ATL-patients but not with anti-HTLV-III-positive sera. Recently, we infected HTLV-I-carrying MT-4 cells with HTLV-III and provoked strong cytopathic effects. This system enabled testing for neutralizing antibodies to HTLV-III. Neutralizing titers to HTLV-III of five anti-HTLV-III-positive sera ranged from 1 : 720 to 1 : 9,000. In contrast, all five seronegative controls showed no or only low reactivity to HTLV-III envelope (1 : 80 and 100). However, three out of five anti-HTLV-I-positive sera exhibited weak cross-reactivities with HTLV-III. The reactivities were expressed as less than 1 : 160, except for one case (1 : 720). They were considered to be nonspecific since they were negative for HTLV-III antibodies in the radioimmunoprecipitation studies.     

The trans-activator gene of the human T cell lymphotropic virus type III is required for replication

The trans-activator gene (tat-III) of the human T lymphotropic virus type III (HTLV-III/LAV) is shown to regulate positively the expression of viral proteins. Viruses in which the tat-III gene is deleted are incapable of prolific replication and do not demonstrate cytopathic effects in T4+ cell lines. These defects can be fully complemented in cell lines that constitutively express the tat-III gene product. We conclude that the tat-III gene product is required for efficient replication of HTLV-III in T4+ cells, and for that reason is important for the cytopathic effects of virus infection. These observations predict that inhibitors of the tat-III gene product may constitute effective therapeutic agents.     

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.     

Induction of anti-HIV neutralizing antibodies by synthetic peptides.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T-lymphotropic virus (HTLV) type III (HTLV-III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV-III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV-III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).     

Human retroviral env and gag polypeptides: serologic assays to measure infection

Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV-infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome.     

Budding process and fine structure of lymphadenopathy-associated virus (LAV)

The budding process and fine structure of lymphadenopathy-associated virus (LAV), were studied by indirect immunofluorescence (IF) and electron microscopy (EM). By IF, LAV antigen was seen to be distributed focally within infected CCRF-CEM cells. Consistent with this finding, electron micrographs showed that LAV particles occurred in a focally aggregated state in a restricted area of the surface of the infected cells. LAV particles possessed bar-shaped, dense and central or eccentric cores. In addition, two or more cores were occasionally observed in one virus particle, or the cores were sometimes absent when thin sections were examined. The envelope of the virus particles had an irregular structure, although LAV particles were approximately spherical.     

Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III.

The human T-cell lymphotropic virus type III (HTLV-III/LAV) is a retrovirus associated with acquired immune deficiency syndrome. The region on the viral genome that is necessary for trans-activation of the HTLV-III/LAV long terminal repeat called tatIII has previously been determined to lie between nucleotides 5365 and 5607. Here we report that a bacterial fusion protein containing amino acid sequences specified by the first coding exon of the tatIII gene is recognized by some patient antisera. We also demonstrate that lymphoid and epithelial cells that express the trans activator function express a 14-kilodalton (kDa) protein recognized by a patient antiserum that reacts with the bacterial tatIII fusion protein. Cells transiently transfected with a deletion mutant of the trans activator protein produce a 12-kDa protein rather than the 14-kDa protein. These observations indicate that the tatIII region contains a functional gene and is capable of expressing a protein that migrates with an apparent molecular size of 14 kDa in some lymphoid and epithelial cells transfected with plasmids containing the tatIII region. We propose that the product of the trans activator gene be designated p14tat-III.     

Polymorphism of the 3'' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.