Attenuation of human immunodeficiency virus type 1 cytopathic effects by replacing a 424-bp region of envelope from a noncytopathic biological clone.

We analyzed the env genes of cytopathic and noncytopathic biological clones derived from two HIV-1-infected children with discordant clinical courses. Chimeric viruses were constructed by switching env regions from V2 through V3 of the biological clones with the corresponding region from the molecular clone NL4-3. These HIV-1 chimeric viruses exhibited similar replication kinetics as well as syncytium-inducing abilities. The chimeric virus containing the env region of noncytopathic biological clone, GC6 8-4, was noncytopathic in an in vitro cell-killing assay, while the chimeric virus containing the env region of cytopathic biological clone, HC4, was cytopathic in the in vitro cell-killing assay. These studies suggest the presence of a cytopathicity determinant that maps to the envelope sequences contained within the downstream region of V2 and within the V3 region (nucleotide position 6822 to nucleotide position 7250, based on NL4-3 sequence).

In vitro characterization of HIV type 1 biological clones from asymptomatic and symptomatic pediatric patients.

To investigate the mechanisms of HIV-1 cytopathogenicity, functional biological HIV-1 clones were isolated from two infected children with high viral loads in vivo. Clone HC4 was isolated from a symptomatic child and clone GC6 8-4 was isolated from an asymptomatic child. These clones were characterized for their ability to induce syncytia, and to replicate and induce single-cell death in peripheral blood-derived normal CD4 T cell cultures containing anti-CD4 antibody. Despite similar viral loads as determined by p24 antigen production or viral RNA expression, GC6 8-4 was noncytopathogenic and HC4 was cytopathogenic. Since we had demonstrated that mitochondrial dysfunction correlated with HIV-1-induced cell death, we determined whether the cytopathogenic HC4 clone decreased mitochondrial viability using a mitochondrial-specific dye, rhodamine-123. Following infection, mitochondrial viability decreased in cells infected with HC4 by day 4 and continued to decline through day 7 when compared to uninfected cells. By day 7 postinfection, greater than 80% of the cells in culture were dead. Similar analyses on CD4 T cells infected with the noncytopathogenic GC6 8-4 demonstrated that mitochondria remained functionally viable and > 90% of the cells excluded trypan blue. These studies describe a cell culture system to study single-cell death in the absence of syncytia and secondary infection. Results with two patient-derived HIV-1 biological clones suggest that loss of mitochondrial viability may play a role in HIV-1-induced cytopathogenicity.

Non-syncytium-inducing HIV type 1 isolated from infected individuals replicates in MT-2 cells.

Human immunodeficiency virus type 1 (HIV-1) isolates from six infected individuals less then 4 years of age were phenotyped for their syncytium-inducing (SI) ability in MT-2 cells. Three viral isolates that induced syncytia were detected. One SI isolate was from an individual who was in disease stage P2A,B,C and two SI isolates were recovered sequentially from another individual who switched from disease stage P1B to P2F. Non-syncytium-inducing (NSI) isolates were detected in two individuals who were in stage P1B of disease, and in a third individual who was in stage P2A of disease. Three sequential isolates obtained over a 2-year period from a fourth individual who progressed from disease stage P1B to P2A,B,C and subsequently died of AIDS-related disease were also found to have the NSI phenotype. To test whether NSI isolates can replicate in the absence of syncytium formation, we analyzed NSI-infected MT-2 cells for production of viral p24 antigen and expression of viral RNA by in situ hybridization. By day 12 postinfection, 6 of 7 NSI viral isolates produced 7- to 36-fold increases in p24 antigen compared to day 6, and expressed viral RNA in 13-20% of cells. A single NSI isolate that did not replicate in MT-2 cells was obtained from an individual who was asymptomatic (stage P1B). The individual rapidly progressed to symptomatic stage P2F and two sequential SI viruses were isolated. These SI isolates replicated in MT-2 cells and induced cytopathic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-arthritic effects demonstrated by an interleukin-2 receptor-targeted cytotoxin (DAB486IL-2) in rat adjuvant arthritis.

DAB486IL-2 is an interleukin-2 receptor-specific cytotoxin which selectively targets and kills cells which bear the high-affinity form of the IL-2 receptor. Since elimination of activated T lymphocytes may be useful in the treatment of rheumatoid arthritis, the effect of DAB486IL-2 treatment in an animal model of arthritis was investigated. We demonstrated that rats treated with DAB486IL-2 during the induction phase of disease have delayed onset of symptoms and significantly reduced severity of inflammation as well as a depressed proliferative response to mycobacterial stimulation in vitro. In addition, the presence of preexisting antibodies to the molecule had no impact on the anti-arthritic effects observed in this model. These data suggest that DAB486IL-2 may have therapeutic potential in the treatment of rheumatoid arthritis.

Impact of interleukin-2-receptor-targeted cytotoxins on a unique model of murine interleukin-2-receptor-expressing malignancy.

DAB486IL-2 is a genetically engineered fusion protein consisting of a portion of diphtheria toxin fused to human IL-2. It is specifically cytotoxic for tumor cells which bear high-affinity IL-2 receptors (IL-2R). DAB389IL-2 is a similarly constructed hybrid protein which is smaller than DAB486IL-2 and is slightly more potent in vitro. We have developed a murine model of IL-2R-expressing malignancy to study the in vivo efficacy of these genetically engineered cytotoxins. Following intravenous administration of CP3 cells, C57BL/6 mice develop tumors which are lymphatic in distribution. When mice are injected i.v. with 10(6) CP3 cells, 90% of the animals show signs of observable tumor by day 10 to 20; death occurs in 50% of untreated animals by day 30. Intravenous treatment of mice with DAB486IL-2 (10 micrograms daily for 10 days), beginning 24 hr after administration of CP3 cells, increases mean survival time by approximately 50%. In comparative studies, DAB389IL-2 is more potent in vivo than DAB486IL-2, with approximately 90% of treated animals with no evidence of tumor at 60 days. The mechanism of action of tumor inhibition by DAB486IL-2 is specific, since treatment of animals which have IL-2R-negative EL4 tumors has not resulted in increased survival time. In addition, treatment of such tumors with DAglu53B486IL-2, a fusion protein which can bind to the IL-2R but is incapable of inhibiting protein synthesis, is ineffective.