Ambros W. Hügin

Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein

The objective of this study was to identify and characterize the gene encoding a protein of approximately 12 kDa that is secreted from cells infected with the vaccinia virus. The absence of this protein from the medium of cells infected with a spontaneous deletion mutant (6/2) suggested that the open reading frame (ORF) was located within a 12,800-base pair segment near the left end of the genome (G. Kotwal and B. Moss, Nature (London) 335, 176-178, 1988). Antibody to the 12-kDa protein immunoprecipitated an appropriate size in vitro translation product of mRNA that hybridized to a DNA segment containing an ORF (N1L) that could encode a 13.8-kDa polypeptide. The similarity in the sizes of the in vitro translation product and the secreted protein was consistent with the absence of processing. Transcriptional analysis revealed major and minor early RNA start sites preceding the N1L ORF as well as a late RNA start site with an atypical TAAAAT sequence. The N1L gene was interrupted by replacing a segment of the ORF with the Escherichia coli beta-galactosidase gene. When two-dimensional polyacrylamide gel electrophoretic patterns of [35S]methionine-labeled proteins secreted from cells infected with parental and recombinant viruses were compared, a spot missing from the latter corresponded in molecular weigh and isoelectric point with that predicted from the N1L ORF. The latter analysis revealed the presence of other secreted proteins of similar molecular weight but different isoelectric points that also appear to map within the left end of the vaccinia genome. The recombinant virus was attenuated as judged by the increased intracranial LD50 for mice but nevertheless induced antibody and cytotoxic responses after intradermal and intraperitoneal injections. Relative to the parental virus, the recombinant was also more attenuated for immunodeficient nude mice, based on their survival time after infection.

Roles of vaccinia virus in the development of new vaccines

Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines. For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.

Variability in the growth sustaining capacity of medium batches.

Medium batches, analysed in various spontaneous and mitogen induced proliferation assays, revealed heterogeneity in their growth promoting activity. This can critically affect test results and suggests that culture media can be a source of variability and problems in cell culture work. The manufacturers of media should broaden their quality control of medium batches.

Murine AIDS Induces Viremia and Functional and Phenotypic Alterations in Blood Cells

Background: Murine acquired immunodeficiency syndrome (MAIDS) is characterized by generalized lymphoproliferation and progressive immunodeficiency. It is induced by a mixture of two replication-competent murine leukemia viruses (MuLV) and a disease-causing, replication-incompetent defective MuLV. Infection leads to specific phenotypic and functional alterations of lymphocytes in lymphoid organs. Methods: We analyzed phenotypic, virological and functional parameters in the blood of mice infected with MAIDS virus. Results: Disease progression correlated with increasing viremia, a loss of mitogen responsiveness of T lymphocytes, and the appearance of CD4+ Thy1– T lymphocytes. At >9 weeks after infection, the distribution of leukocyte cell populations became very heterogeneous, and late-stage leukemic events were observed in 5 of 23 mice. Conclusions: Virus titers, mitogen responsiveness and the presence of CD4+ Thy1– T lymphocytes can efficiently be monitored in the blood and serve as diagnostic parameters to monitor disease progression. Acute leukemic events occurring at the terminal stage could be responsible for the death of at least some of the mice with MAIDS.