FIG. 3.

Breadth of rabbit anti-gp120 IgG responses in rabbit sera showed positive neutralizing antibody responses against JR-FL. (A) Cross-reactivity of antibodies specific for HIV-1 gp120s in rabbit sera 2 weeks after the second protein boost was assessed using a Biacore 3000 biosensor. Each serum sample was diluted 1/100 in HBT-CMD running buffer (HEPES buffer, pH 7.4, 150 mM NaCl, 0.1% Tween 20, 0.5% soluble carboxymethyl dextran) and passed at a flow rate of 5 μl/min for 20 min over surfaces to which the indicated gp120s had been covalently coupled to a density of approximately 750 relative units (RU). A reference flow cell was coated with alcohol dehydrogenase to approximately the same density to control for nonspecific surface effects. The response obtained from each sensorgram 5 s prior to the end of the association phase was determined. The response from preimmune serum of each rabbit was subtracted to control for nonspecific binding of serum proteins to the sensor surface. (B) The same rabbit serum samples at 2 weeks after the second protein boost were analyzed for neutralizing activities against four primary HIV-1 isolates (1196, 0692, 6101, and PVO) in addition to the autologous JR-FL strain. All of the viruses were grown in peripheral blood mononuclear cells, and the assays were conducted in 5.25.EGFP.Luc.M7 cells (17) with rabbit serum at a 1:10 dilution. Neutralizing activities are presented as the percent reduction of RLU compared to virus control wells after subtraction of background RLU. Preimmune sera for all four rabbits did not show any positive neutralizing activities (data not shown).