Sigrid Nick

Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus

Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation.

Potential significance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC) in immunized and infected rhesus macaques

The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAC) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4+ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4+ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAC proteins were protected from severe thrombocytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAC antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.

Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.

We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.

Virus-specific cytotoxic T-lymphocytes in HIV-2-infected cynomolgus macaques

ObjectiveSpecific cytotoxic T-lymphocytes (CTL) are induced in humans or monkeys after infection with HIV-1 or SIVmac, respectively. Since, like HIV-1, HIV-2 causes AIDS, our objective was to determine the characteristics of the HIV-2-specific CTL response. DesignSince it is rarely possible to study cellular immunity in individuals, because of the small number of HIV-2-infected patients available in Europe and the necessity for co-operation in the performance of sequential CTL assays, cynomolgus macaques were infected with HIV-2. Autologous transformed B-lymphoblastoid cell lines infected with recombinant vaccinia viruses were used as target cells for cytotoxicity assays. MethodsRecombinant vaccinia viruses expressing HIV-2 genes were constructed to infect B-lymphoblastoid cell lines from macaques. These cells were used as target cells for cytotoxicity assays with peripheral blood mononuclear cells from HIV-2BEN-infected cynomolgus macaques. To characterize the effector cells, CD8+ cells were separated with immunomagnetic beads. Major histocompatibility complex (MHC) restriction of the cytotoxic cells was determined by incubation with matched or mismatched target cells. ResultsHIV-2BEN-infected cynomolgus macaques raised CTL against proteins of the three major viral structural genes, gag, pol and env. The cytotoxic cells were CD8+ and their activity was MHC class I-restricted. In contrast to SIVmac-infected macaques, env-specific lysis was mediated exclusively by CD8+ cells. CTL from individual animals recognized different viral proteins and the recognition pattern varied over time. ConclusionsLike HIV-1 and SIVmac, HIV-2 induces virus-specific CTL. The variation of antigen recognition between individual animals and over time indicates that sequential experiments are necessary to determine the complete spectrum of the CTL response of infected animals. HIV-2-infected macaques represent a suitable model for investigations into the cellular immune response against HIV.

Molecular and biological characteristics of a novel HIV-2 isolate, HIV-2HOM

In 1991 a new HIV-2 isolate (HIV-2HOM) was isolated first from a German individual most likely infected in West Africa in the beginning of the 1970s. The virus was isolated from both, the plasma and the peripheral blood lymphocytes of the patient by using OKT-3-stimulated cord blood lymphocytes. The recovered viruses could be further propagated on Jurkat cells and exhibited a broad cell tropism. Biochemical and antigenic properties of HIV-2HOM were examined by radioimmunoprecipitations. For a more detailed molecular characterization, a 1520 bp DNA fragment from the env gene and a 722 bp DNA fragment from the pol gene were amplified by polymerase chain reactions, cloned and sequenced. A comparison of both sequences to prototypic HIV-2 and SIV isolates revealed a close relationship to HIV-2ST. This strain originated from an asymptomatic Senegalese individual and is supposed to be of reduced pathogenicity. Taking into account genetic data, it may be assumed that HIV-2HOM and HIV-2ST are closely related strains with different growth characteristics and pathogenic features.