Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin.

Abstract

An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for attenuated HIV, which could be used as a vaccine. We have reconstructed the mutation found in the E variants within the infectious HIV clone HXB-2 and demonstrated that the resulting virus retains its noninfectious phenotype.

Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells

“…Western blotting (immunoblotting) was performed with cell lysates and HIVIG to detect HIV-specific antigens (16). Immunoperoxidase staining was performed on ethanol-permeabilized cells with HIVIG (19). Reverse transcriptase (RT) activity was measured in cell supernatants with a poly(rA) template and radiolabeled dTTP (10,19).…”

“…We have previously selected persistently infected cell lines for variants with an anti-gp120 immunotoxin. We have shown that the variants fell into two general classes: those in which only the expression of HIV Env (envelope proteins gp160, gp120, and gp41) was altered and those in which the production of all viral proteins was diminished or absent (19). Altered Env expression was shown to be due to a mutation in gp41, resulting in a truncated form of the transmembrane protein and altered processing of both the envelope protein and the intact virus.…”

“…Upon entry into cells expressing the target antigens at their surface, the immunotoxins kill the cells, primarily through ribosomal inactivation. Because the target antigen must be expressed on the cell surface and then internalized to obtain cell killing, the immunotoxins are useful as probes of HIV variation and to study the cell biology of HIV infection (15,16,19).…”

Immunotoxins

“…Issues of specific concern with anti-HIV immunotoxins include variability of clinical isolates of the virus, evolution of immunotoxin-resistant variants, [39] the presence of anti-HIV antibodies that may block the immunotoxin from reaching its target,rlO] and the lack of surface expression of HIV proteins on certain cell types. In addition, the secretion of soluble gp120 and virus-associated gp 120 and gp41 may prevent the immunotoxin from reaching the target cell.…”

Group B streptococcal opacity variants

“…The fixed colonies were pelleted and washed twice with PBS containing 0.2 M sucrose and then twice with tap water. The samples were stained with aqueous uranyl acetate and fixed as described previously (17). To determine if there was a difference in the thicknesses of the extracellular matrix (cell wall, capsule, etc.)…”