Ligand-mediated cytolysis of tumor cells: use of heregulin-zeta chimeras to redirect cytotoxic T lymphocytes.

New specificities may be engrafted onto lymphocytes by the transfer of genes for chimeric receptors that combine antigen recognition and signal-transducing elements. We have engineered and evaluated a new class of chimeric receptors that use the natural ligands of receptors found to be frequently overexpressed by cancer cells. The heregulin molecule, a ligand for Her3 and Her4 receptors when fused with the CD3 zeta-chain, was capable of redirecting T lymphocytes to recognize and respond to cancer cell lines that overexpress these receptors. Thus, CD8+ T lymphocytes were isolated from a healthy individual and transduced to express the chimeric heregulin-zeta receptor. These modified effector cells acquired the ability to specifically lyse a breast cancer cell line that overexpresses Her3 and Her4. A new class of chimeric receptors, such as heregulin-zeta, endowing anti-cancer effector cells with the potential to recognize and eliminate tumor targets, are likely to increase the effectiveness of adoptive immunotherapy for the treatment of cancer.

Expression of Fas/CD95 and Bcl-2 by primitive hematopoietic progenitors freshly isolated from human fetal liver.

The cell-surface expression and the functional status of the CD95/Fas antigen on primitive hematopoietic progenitors (PHPs) freshly isolated from human fetal liver (FL) were studied. PHPs were phenotypically defined as CD34++ CD38 -/+ cells. The most immature subfractions of PHPs, CD34++CD38- and CD34+2CD38+ FL cells, expressed CD95, whereas the more mature CD34++CD38++ and CD34+CD38++2 FL cells displayed low CD95 expression. Combinations of cytokines, such as kit ligand (KL) + interleukin-3 or KL + granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated the expression of CD95 on PHPs upon in vitro culture. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) further increased the CD95 expression induced by KL+GM-CSF. The hematopoietic potential of sorted CD34++lineage (lin)- CD95+ versus CD34++ lin-CD95-FL cells was compared by colony-forming unit-culture (CFU-C) assays performed in serum-deprived medium. Lin+ cells were composed of erythrocytes, monocytes, T cells, B cells, and natural killer cells. Our results indicated that both CD95- and CD95+ subsets contained pluripotent progenitors, generating myeloid and erythroid progenitors. The functional status of CD95 and the effects of TNF-alpha and IFN-gamma, cytokines known to induce CD95-mediated apoptosis, were analyzed by incubation of PHPs in the presence of anti-CD95 monoclonal antibodies (MoAbs). The effect of anti-CD95 MoAbs was measured by viable cell counting, flow cytometry, and CFU-C assays. A decrease of CFU-C numbers was observed in the presence of anti-CD95 MoAbs and TNF-alpha and/or IFN-gamma. However, whereas growth factor deprivation induced apoptosis of PHPs, cross-linking of CD95 did not lead to apoptosis of PHPs measured by flow cytometry and viable cell counting. The correlation of increased intracytoplasmic levels of bcl-2 with high levels of cell-surface CD34 and the presence of CD95 on fresh FL cells suggests that bcl-2 may be involved in protecting against CD95-mediated apoptosis of FL PHPs.

Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.

An epitope on the surface envelope glycoprotein (gp130) of simian immunodeficiency virus (SIVmac) involved in viral neutralization and T cell activation.

SIVmac infection of macaques is an important animal model for HIV infection and AIDS; this model is being utilized for development of antiviral therapies and vaccines. In the present article, we sought to identify neutralization epitopes of SIVmac envelope surface glycoprotein (gp130). Algorithms were used to predict antigenicity of specific regions. Four regions from the primary amino acid sequence of the viral surface glycoprotein were selected. A synthetic peptide representing one of these regions (414-434) induced virus-neutralizing antibodies in mice; in addition, this peptide induced T cell-proliferative responses in macaques. To address the in vivo relevance of these observations, we demonstrated that experimentally infected macaques produce antibodies to the neutralization epitope. In addition, rhesus macaques protected against infection by an inactivated SIV vaccine develop antibodies that bind to peptide 414-434. These observations demonstrate that the region that includes the sequence 414-434 in the fourth variable domain (V4) of SIVmac gp130 contains both a linear neutralization epitope and a T cell epitope.

SIV envelope glycoprotein epitopes recognized by antibodies from infected or vaccinated rhesus macaques.

We analyzed SIV-specific monkey sera to localize B-cell epitopes of the envelope glycoprotein of SIV (gp130), using overlapping synthetic peptides representing the entire SIV gp130 protein and sera from experimentally infected monkeys and monkeys immunized with whole, inactivated SIV. A B-cell epitope which induces neutralizing antibody production and T-cell responses was characterized as well as a new B-cell epitope and a previously described neutralizing epitopes. Vaccinated monkey sera recognize the three epitopes differentially relative to unimmunized controls, and a correlation appears to exist between degree of cross-neutralization by infected monkey sera and degree of binding to these three regions.

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac.

Simian immunodeficiency virus (SIV), a lymphocytopathic lentivirus, induces an AIDS-like disease in rhesus macaques (Macaca mulatta). A pathogenic molecular clone of rhesus macaque SIV (SIVmac), SIVmac-239, replicates and induces cytopathology in T lymphocytes but is restricted for replication in macrophages. In contrast, a nonpathogenic molecular clone of SIVmac, SIVmac-1A11, replicates and induces syncytia (multinucleated giant cells) in cultures of both T lymphocytes and macrophages. SIVmac-1A11 does not cause disease in macaques. To map the viral determinants of macrophage tropism, reciprocal recombinant genomes were constructed between molecular clones of SIVmac-239 and SIVmac-1A11. Infectious recombinant viruses were rescued by transfection of cloned viral genomes into permissive lymphoid cells. Analysis of one pair of reciprocal recombinants revealed that an internal 6.2-kb DNA fragment of SIVmac-1A11 was necessary and sufficient for both syncytium formation and efficient replication in macrophages. This region includes the coding sequences for a portion of the gag gene, all of the pol, vif, vpr, and vpx genes, the first coding exons of tat and rev, and the external env glycoprotein gp130. Thus, the transmembrane glycoprotein of env, the nef gene, the second coding exons of tat and rev, and the long terminal repeats are not essential for in vitro macrophage tropism. Analysis of additional recombinants revealed that syncytium formation, but not virus production, was controlled by a 1.4-kb viral DNA fragment in SIVmac-1A11 encoding only the external env glycoprotein gp130. Thus, gp130 env of SIVmac-1A11 is necessary for entry of virus into macrophages but is not sufficient for a complete viral replication cycle in this cell type. We therefore conclude that gp130 env and one or more genetic elements (exclusive of the long terminal repeats, transmembrane glycoprotein of env, and second coding exons of tat and rev, and nef) are essential for a complete replication cycle of SIVmac in rhesus macaque macrophages.

In vitro macrophage tropism of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

The significance of infection of mononuclear phagocytes by immunodeficiency lentiviruses of primates is not clearly established. To explore the relationship of macrophage tropism and pathogenesis, conditions to culture and infect monocyte-derived macrophages from rhesus macaques were established and the growth properties of two molecular proviral clones of simian immunodeficiency virus (SIVmac) were studied. Rhesus macrophages supported productive infection of the nonpathogenic SIVmac-1A11 clone; extensive cytopathology characterized by formation of multinucleated giant cells and release of particle-associated reverse transcriptase activity in culture supernatant were observed. In contrast, the pathogenic SIVmac-239 did not establish a productive infection of macrophages and no cytopathology was observed. Both SIVmac-1A11 and SIVmac-239 replicated and induced cytopathic effects in cultures of rhesus peripheral blood lymphocytes and the Cemx174 lymphoid cell line. These results, together with the previously published reports on the pathogenic potential of these two clones of SIVmac, suggest that macrophage tropism measured in vitro does not correlate with in vivo virulence.

Rhesus macaques inoculated with molecularly cloned simian immunodeficiency virus.

We have isolated a biologically active molecular clone of simian immunodeficiency virus (SIV), SIVmac 1A11, originally obtained from a rhesus macaque at the New England Regional Primate Research Center. Virus derived from cells transfected with this clone is cytopathic for rhesus peripheral blood mononuclear cells, replicates in cultures of rhesus macrophages, and infects rhesus macaques when inoculated intravenously. Six macaques inoculated with SIVmac 1A11 all became infected and produced antibodies to viral envelope glycoproteins that neutralized virus. Antibodies to viral core proteins were detected in only one animal. No clinical signs of disease were observed throughout 7 months postinoculation.

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus.

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.

Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein.

Formation of syncytia, with progression to cell death, is a characteristic feature of in vitro cultures of susceptible cells infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). Viral antigen-positive multinucleated giant cells have also been observed in histological sections from infected individuals. In vitro, formation of these multinucleated giant cells occurs through cell fusion which is dependent on cell-surface expression of the differentiation antigen CD4. Utilizing a recombinant vaccinia virus containing the gene for the envelope glycoprotein of HTLV-III/LAV, we demonstrate that cell-surface expression of this protein, in the absence of other HTLV-III/LAV structural or regulatory proteins, is sufficient to induce CD4-dependent cell fusion, leading to cell death, one of the characteristic manifestations of AIDS (acquired immune deficiency syndrome) virus cytopathology. This process may contribute to the loss of CD4+ T cells seen in AIDS.

The AIDS-associated retrovirus is not sensitive to lysis or inactivation by human serum.

Unheated human serum does not lyse the AIDS-associated retrovirus (ARV), change the density of the virus, or suppress its ability to infect human peripheral mononuclear cells. The results indicate that ARV and the human oncovirus HTLV-I, unlike other animal retroviruses, are resistant to the effect of human serum. These two human viruses coming from different retrovirus subfamilies may be pathogenic because of this lack of sensitivity to human complement components.

Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions.

The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA)oligo(dT) and prefers Mg2+ over Mn2+ as a divalent cation. It can use other template-primer combinations but with poly(rCm)oligo(dG), it prefers Mn2+ over Mg2+. Detection of ARV reverse transcriptase in culture fluids is substantially increased with an optimal KCl concentration and a special combination of EGTA and reducing agents. Our results indicate some distinguishing characteristics of the ARV reverse transcriptase and optimal conditions for its detection.