Development of a novel allo-independent HIV-1 virus preparation for use in immunoassays.
The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.
Department of Medical Microbiology, University of Manitoba, 539-730 William Avenue, Winnipeg, MB, Canada.
Sandra
S
Koesters
W
W
Rutherford
Keith R
KR
Fowke
D018929:Cell Culture Techniques / Q000379:methods; D002474:Cell-Free System / Q000276:immunology; D002478:Cells, Cultured; D015693:Gene Products, nef / Q000096:biosynthesis / Q000235:genetics; D015488:HIV Antigens / Q000235:genetics / Q000276:immunology*; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000235:genetics; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006680:HLA Antigens / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D020014:K562 Cells / Q000276:immunology / Q000821:virology; D014162:Transfection; D019076:Transgenes; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D015488:HIV Antigens; D016655:HIV Core Protein p24; D015699:HIV Envelope Protein gp120; D006680:HLA Antigens; D006684:HLA-DR Antigens; D015395:Histocompatibility Antigens Class I; D054311:nef Gene Products, Human Immunodeficiency Virus