A simple, rapid, quantitative syncytium-forming microassay for the detection of human immunodeficiency virus (HIV-I) isolates is described. A virus-syncytial sensitive clone of CEM cells (CEM-SS) was identified and made adherent to flat bottom 96-well microtiter dishes. Following the addition of virus, these cells develop easily quantifiable, adherent syncytia on a background of confluent, normal CEM-SS monolayer in 4 to 6 days. One-hit kinetics for syncytia formation were obtained at various multiplicities of infection. Syncytia are associated with complete virion production and cytoplasmic localization of the p24 core protein (detected by immunofluorescence). Total infectious virus can be accurately determined in this assay; these results showed a close correlation with p24 and gp120 induction when microtiter well supernatants were passed to fresh cells and evaluated by competitive radioimmunoassay. Studies of p24 antigen induction at and beyond the end point of syncytia formation indicate that there are no detectable nonsyncytial variants in standard HIV-I stocks. Six divergent HIV-I isolates (HTLV-IIIB, -RFII, -MN, -RUTZ, -CC, and LAV-1), as well as HTLV-IIIB and LAV-1 reisolated from persistently infected chimpanzees, produce quantifiable syncytia which vary slightly in their developmental morphology. Accurate neutralization titers are readily obtained from easily constructed multiplicity curves derived from serial dilutions of test sera. Inherent within this system is a flexible method for studying various kinetics of antibody/virus interactions, as well as blocking and interference studies with any candidate antiviral compounds.

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IUB gpl20-vaccinated and-challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 MB common nonapeptide (IQRGPGRAF) derived from the gpl20 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IUB monoclonal antibody (0.5 1) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralizationresistant isolates consisted of the lower-replication-competent virus subpopulation of the HIV-1 IHIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gpl20 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gpl20s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gpl20 were then inoculated with purified RF gpl20. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gpl20 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.