Expression of AIDS virus envelope gene in recombinant vaccinia viruses

Acquired immune deficiency syndrome (AIDS) is an infectious disease characterized by severe impairment of the patient's cell-mediated immune system. Several lines of evidence have indicated that the aetiological agent of AIDS is a group of T-lymphotropic retroviruses, variously known as lymphadenopathy-associated virus (LAV), human T-lymphotropic virus type III (HTLV-III) and AIDS-associated retrovirus (ARV). Serological surveys have indicated that as many as one million people in the United States may have been infected by LAV/HTLV-III, and the spread of AIDS has become a global concern. The need for a better understanding of the viral immunology and for a vaccine against AIDS is self-evident. To this end, we have constructed recombinant vaccinia viruses containing the envelope (env) gene of LAV, and demonstrate here that cells infected with these viruses express immunoreactive proteins similar to those present on LAV virions. Experimental animals infected with these recombinant viruses elicited antibodies that specifically recognized LAV envelope proteins.     

Expression of the HTLV-III envelope gene by a recombinant vaccinia virus

The discovery that the aetiological agent of acquired immune deficiency syndrome (AIDS) is a retrovirus, referred to as human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) (for review see ref. 1), has raised the possibility of developing a vaccine. In this regard, the envelope (env) proteins of murine retroviruses can induce protective immunity in mice. The HTLV-III env gene specifies a primary polypeptide of approximately 860 amino acids that is glycosylated to form a precursor of relative molecular mass (Mr) 160,000 (gp160), which gives rise to mature membrane-associated proteins of Mr 120,000 (gp120) and 41,000 (gp41). The HTLV-III env gene has been expressed in Escherichia coli and by simian virus 40 (SV40) vectors but formation of the authentic proteins has not been demonstrated. Here, we describe the expression of the complete env gene by a vaccinia virus vector. Evidence is presented that synthesis, glycosylation, processing and membrane transport of the env polypeptide occurred without other HTLV-III gene functions; the env protein was recognized by sera from unrelated AIDs patients; and a single vaccination with the infectious recombinant vaccinia virus induced antibodies to gp120 in mice.     

A molecular clone of HTLV-III with biological activity

Acquired immune deficiency syndrome (AIDS) is an epidemic immunosuppressive disease characteristically associated with a depletion of T lymphocytes of the helper/inducer phenotype. Numerous converging lines of research have implicated a human T-cell lymphotropic retrovirus, HTLV-III, in the pathogenesis of AIDS. Recently, several distinct forms of the HTLV-III genome were molecularly cloned in phage and extensively characterized. In the present study, a clone containing full-length HTLV-III proviral DNA was inserted into a plasmid and used to transfect cord blood T cells from normal newborn humans. We demonstrate that this molecular clone is infectious in vitro and causes marked cytopathic effects on T-cell cultures. This is the first direct evidence that the HTLV-III genome, rather than a minor component of the virus complex, is cytopathic for T cells. Using this biologically competent clone and mutants derived from it, it should now be possible to localize the subgenomic regions that contribute to the biological effects of HTLV-III.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.     

An HTLV-III peptide produced by recombinant DNA is immunoreactive with sera from patients with AIDS

Human T-cell lymphotropic retrovirus type III (HTLV-III), also called lymphadenopathy-associated virus (LAV), has been identified as the aetiological agent of acquired immune deficiency syndrome (AIDS). The sera of most patients with AIDS or AIDS-related complexes, and of asymptomatic individuals infected with HTLV-III, contain antibodies against antigens of HTLV-III. The characterization of these antibodies and their corresponding viral antigens is important not only for understanding immunity against HTLV-III and the pathology of AIDS, but also for the development of diagnostic methods and preventive vaccine for AIDS. Following the successful establishment of a long-term T-cell line permissive for HTLV-III replication, large quantities of virus have been produced, facilitating the purification of viral proteins and the development of mouse monoclonal antibodies against several viral antigens. More recently, the structure of HTLV-III proviral DNA has been elucidated. We now report the production, by genetic engineering methods, of a peptide encoded by a gene segment of HTLV-III. A 1.1-kilobase (kb) EcoRI DNA segment from an isolate of HTLV-III was inserted into a lpp and lac promoter-coupled expression vector, pIN-III-ompA. Escherichia coli transformants of this plasmid produced a peptide of relative molecular mass (Mr) 15,000 (15K) which was strongly immunoreactive with anti-HTLV-III antibodies present in sera from AIDS patients. Lysates of the clones expressing this 15K peptide inhibited the reactivity of the p31 virion protein with AIDS sera, suggesting that it is a fragment of the viral p31 protein. The peptide reacted with sera from all 20 AIDS patients but none of the 8 normal controls tested. These results suggest that the peptide may be useful for detecting anti-HTLV-III antibodies in blood samples.     

Molecular cloning of lymphadenopathy-associated virus

Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1-9.2 kilobases).     

Sequence of simian immunodeficiency virus and its relationship to the human immunodeficiency viruses

The characterization of HIV-1 (HTLV-III/LAV), the human retrovirus associated with AIDS (acquired immune deficiency syndrome) has led to the identification of a group of related human and simian retroviruses which also infect CD4-bearing T lymphocytes. Simian T-lymphotropic virus type III (simian immodeficiency virus) from macaques (STLV-IIIMAC) induces symptoms similar to those of AIDS in infected macaques, but isolates from African green monkeys (STLV-IIIAGM) and mangabeys (STLV-IIMM) appear to be non-pathogenic in these animals. A human virus immunologically related to STLV-IIIAGM (HTLV-IV), reported to have been isolated from healthy humans, has been shown to be almost identical to STLV-IIIAGM, which has called into question the independent origin of these viruses. Here we report the complete DNA sequence of STLV-IIIAGM and analyse its relationship with the genomes of the HTLV-IIIB strain of HIV-1, HIV-2ROD (previously called LAV-2) and several ungulate lentiretroviruses. STLV-IIIAGM and HIV-2 are closely related, and more distantly related to HIV-1.     

Aetiology of AIDS--antibodies to human T-cell leukaemia virus (type III) in haemophiliacs

Human T-cell leukaemia virus type III (HTLV-III) is suspected of having a key role in the pathogenesis of acquired immune deficiency syndrome (AIDS). Epidemiological data suggest that AIDS is transmitted by an infectious agent through intimate contact with body secretions, blood or blood products. To maintain haemostasis, many haemophiliac patients depend on commercially prepared clotting concentrates made from large multi-donor plasma pools and are thus at increased risk of developing the disease. We report here that, using indirect membrane immunofluorescence and radioimmunoprecipitation with SDS-polyacrylamide gel electrophoresis, we have detected antibodies to HTLV-III in 30 of 47 (64%) asymptomatic haemophiliacs and in all of three haemophiliacs who either had or soon developed AIDS. Of 34 samples drawn before 1984, 18 (53%) were antibody-positive, whereas of 16 samples drawn during 1984, 15 (94%) were positive (P less than or equal to 0.002). These data suggest that exposure to HTLV-III antigens is widespread among asymptomatic haemophiliacs.     

Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity

Acquired immune deficiency syndrome (AIDS) is characterized by marked depletion of the T4+ helper subset of T cells. The aetiological agent of the disease, the human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV), specifically kills T4+ cells in vitro. Part of this specificity for the T4+ population residues in the relative efficiency with which HTLV-III infects these cells, as a result of a specific interaction between the T4 molecule and the virus envelope glycoprotein. In addition, the cytotoxic consequences of HTLV-III replication are dependent on cell type, as certain lymphoid and myeloid cells can be productively infected without notable cytopathic effect. Here we investigate the basis for the specific cytotoxicity of the virus, and report that high-level expression of the HTLV-III envelope gene induces syncytia and concomitant cell death in T4+ cell lines but not in a B-lymphocyte line. Syncytium formation depends on the interaction of envelope-expressing cells with neighbouring cells bearing surface T4 molecules. These results explain, at least in part, the specific cytopathic effect of HTLV-III infections.     

The HIV 'A' (sor) gene product is essential for virus infectivity

The genome of the human immunodeficiency virus (HIV) contains several open reading frames (ORFs) not present in other viruses. The 'A' gene, also known as Q2 P'3, ORF-1(4) or sor5, partially overlaps the pol gene; its protein product has a relative molecular mass of 23,000 (Mr 23K) and is present in productively infected cells. The function of this protein is unclear; mutant viruses deleted in 'A' replicate in and kill CD4+ lymphocyte lines, but the high degree of conservation of the deduced amino-acid sequence in nine different HIV isolates (80%) and the presence of analogous genes in HIV-2 and other lentiviruses suggest that the gene function is an important one. Here we describe a mutant virus deficient in the 'A' gene which produces virion particles normally; however, the particles are approximately 1,000 times less infective than wild type. Transcomplementation experiments partially restore infectivity. The mutant virus spreads efficiently when virus-producing cells are co-cultivated with CD4+ lymphocytes, however, indicating that HIV can spread from cell to cell in a mechanism that does not require the 'A' gene product and probably does not require the production of infective virus particles.     

HTLV-III-neutralizing antibodies in patients with AIDS and AIDS-related complex

The isolation of the human T-cell leukaemia (lymphotropic) virus type III (HTLV-III or lymphadenopathy-associated virus) from cells of many patients with acquired immune deficiency syndrome (AIDS) presented the first evidence that the virus was the aetiological agent of the disease. Subsequent seroepidemiological studies have shown the presence of HTLV-III-specific antibodies in the serum of most patients with AIDS and AIDS-related complex (ARC), and in the serum of many individuals at risk for AIDS. Despite these extensive studies, there are no reports of protective effects of HTLV-III antibodies. In contrast, neutralizing antibodies specific for HTLV-I and -II have been identified previously. Therefore, we investigated whether HTLV-III-exposed individuals possess antibody activities capable of inhibiting viral infection. Here, we report that natural antibodies capable of neutralizing HTLV-III infection of H9 cells were detected in most adults AIDS and ARC patients but in no normal healthy heterosexual controls. Geometric mean antibody titres in ARC patients were double those in AIDS patients, and were even higher in two antibody-positive healthy homosexuals. This suggests that virus neutralizing antibodies may exert an in vivo protective effect. The presence of these antibodies indicates an immunological response to HTLV-III which potentially may be manipulated for therapeutic advantage. The methodology used here will be useful in monitoring future vaccine approaches.     

Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients

Human T-lymphotropic virus type III (LAV, HTLV-III) is aetiologically linked to acquired immune deficiency syndrome (AIDS) and persistent general lymphadenopathy (PGL). Specific radioimmunoassays (RIA), enzyme-linked assays, immunofluorescence assays (IFA) and immunoblotting techniques are being used widely to detect serum antibodies to HTLV-III in infected patients and in those at risk of infection. However, these assays do not functionally identify those antibodies that neutralize the infectivity of the virus. We have used three methods of titrating serum neutralizing factors: inhibition of syncytium induction, neutralization of envelope pseudotypes of vesicular stomatitis virus (VSV) and reduction of infectivity of HTLV-III for a cell line permissive to virus replication. We report here that sera from subjects in various disease categories possess only low-level neutralizing activity, even when antibodies to viral membrane antigens are present in high titre. Envelope pseudotypes prepared from four HTLV-III isolates made in three different countries are equally sensitive to neutralization by positive sera, including sera from patients yielding two of the virus isolates.     

Surface phenotypes in T-cell leukaemia are determined by oncogenic retroviruses

Spontaneous thymic leukaemia in experimental mice is the result of a complex series of genetically controlled events. An important step in this process involves the production by thymocytes of recombinant polytropic retroviruses (MCF viruses). These leukaemogenic agents arise by recombination of genes from the env regions of endogenous precursor viruses. Sequences in these regions encode the envelope glycoprotein gp70 (ref. 6). Thus far, each cloned isolate of recombinant virus from AKR and HRS/J mice has been found to possess unique oligonucleotide sequences in its env region, as well as clone-specific peptides in its gp70 (refs 7,8). Therefore, the polytropic viruses of these leukaemia-susceptible mice are extremely diverse. These findings suggest that random recombination of env genes gives rise to leukaemogenic polytropic viruses. McGrath and Weissman have proposed that thymocytes with cell surface receptors for the gp70 of a particular leukaemogenic virus are the target cells for malignant transformation by that specific virus. In view of the diversity of polytropic viral gp70, their hypothesis would predict extensive phenotypic diversity among spontaneous thymic leukaemias. In contrast, leukaemias induced by a particular leukaemogenic recombinant virus would always have the same phenotype. Here we verify these predictions experimentally.     

A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS

The first experimental immunization of humans against the AIDS retrovirus, HIV-1, was started in a series of HIV seronegative, healthy volunteers in November 1986. For the primary vaccination recombinant vaccinia virus (V25) expressing the complete gp160 env protein of the HTLV-IIIB strain of HIV-1 was introduced by scarification. This elicited a weak primary response which we subsequently attempted to enhance by additional immunizations (boosting), using four different immunization protocols. We report here that intravenous injection of paraformaldehyde-fixed autologous cells infected in vitro with V25 (individual D.Z.) gave the best results. This individual received second and third boosts of intramuscular gp160 derived from an HTLV-IIIB clone using the hybrid vaccinia virus/bacteriophage T7 expression system. An anamnestic humoral and cellular immune reaction was achieved for over one year after the original vaccination, with high levels of antibodies to the viral envelope, and neutralizing antibodies against divergent HIV-1 strains such as HTLV-IIIB and HTLV-IIIRF (also called HTLV-III HAT) after the first boost. In addition, group-specific cell-mediated immunity and cell-mediated cytotoxicity against infected T4 cells were obtained after the primary vaccine and enhanced by the boosts. Finally, skin tests showed both immediate and delayed hypersensitivity to gp160 in vivo. Although this protocol is not practical for a large scale vaccine trial, our results show for the first time that an immune state against HIV can be obtained in man.     

Identification of a protein encoded by the vpu gene of HIV-1

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.