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Adenovirus-polylysine-mediated foreign gene expression in EBV-transformed B-cells (B-LCL): potential for novel CTL target cells.


Int Conf AIDS. 1992 Jul 19-24;8(3):43 (abstract no. PuA 6192). Unique

INTRODUCTION: The most widely used cytotoxic T-cell (CTL) killing assay for the study of HIV-specific immunity employs infection of B-LCL with recombinant vaccinia viruses. This approach may be unsuitable for the study of recipients of vaccinia-derived HIV vaccines owing to the high degree of background vaccinia-specific killing elicited. Furthermore, as vaccinia is lytic for B-LCL, target cells must be prepared fresh for each assay. We sought therefore to develop a vaccinia-independent system that could also allow for the formation of stably transformed target cells. METHODS: Plasmids encoding a firefly luciferase (luc) reporter gene under the control of the Cytomegalovirus (CMV) or Rous Sarcoma virus (RSV) promotors were derived from pCLuc4 or pRSVL, respectively. An RSVL/luc plasmid encoding the Epstein-Barr virus genes EBNA-1 plus OriP was derived, designated 220RSVLuc alpha. Plasmid delta Ehis expresses the HIV-1 gag gene under control of the HIV-1 long terminal repeat. For the gene transfer experiments, adenovirus-polylysine conjugates were formed by linkage of the polylysine moiety to adenovirus by a streptavidin-biotin coupling. Reporter DNA was combined with conjugates to form complexes. Final condensation of DNA was with polylysine (pL) or transferrin-polylysine (hTfpL). RESULTS: Optimal transfection conditions were determined with the luc reporter: 1) 24-48 hour expression time for maximum gene expression, 2) RSV or CMV promotor worked equally well, 3) EBNA-1 and OriP were not essential for transient gene expression and 4) pL was superior to hTfpL for gene expression in most, but not all B-LCL lines. In addition, we have successfully expressed HIV-1 gag in B-LCL in transient assays, but at lower levels than that expressed by recombinant vaccinia virus (4 vs 120 pgm/ml of p24, respectively). CONCLUSION: The adenovirus-polylysine vector system can be utilized to transduce B-LCL cellular target cells. This vector system may overcome some of the shortcomings of the recombinant vaccinia viruses. Importantly, we have used it to induce HIV-1 gag expression successfully in B-LCL. Owing to the presence of a selectable marker on our HIV-1 gag-expressing construct, the establishment of stably transformed target cells for CTL killing assays may be possible with this system as well.

Adenoviridae/*GENETICS Animal Antigens, Viral/GENETICS B-Lymphocytes/IMMUNOLOGY Beetles *Cell Transformation, Viral Cytomegalovirus/*GENETICS Cytotoxicity, Immunologic DNA-Binding Proteins/GENETICS Gene Expression *Gene Expression Regulation, Viral *Genes, gag Herpesvirus 4, Human/*GENETICS/IMMUNOLOGY HIV-1/*GENETICS Luciferase/*GENETICS/METABOLISM *Polylysine Promoter Regions (Genetics) Sarcoma Viruses, Avian/*GENETICS T-Lymphocytes, Cytotoxic/IMMUNOLOGY *Transfection Vaccinia Virus/GENETICS ABSTRACT


Information in this article was accurate in December 30, 1992. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.