Figure 1: Characterization of SHIV 89.6 VLPs. SHIV 89.6 VLPs were produced by coinfection of Sf9 cells with rBVs expressing HIV 89.6 Env and SIVmac239 Gag proteins at the MOI (multiplicity of infection) of 5 and 2, respectively, and purified as described in Section 2. SIV Gag VLPs were produced by infection of Sf9 cells with rBV expressing SIVmac239 Gag proteins and purified similarly. (a) Characterization of SHIV 89.6 VLPs by Western blot. 5  𝜇 g total proteins were taken from each VLP preparation and analyzed by SDS-PAGE followed by Western blot using rabbit-anti-gp120 antibody for detection of HIV Env proteins and monkey-anti-SIV fordetection of the SIV Gag proteins. Lanes 1, SHIV 89.6 VLP; 2, SIV Gag-only VLP. (b) Comparison of HIV 89.6 Env amount in VLP preparations by a quantitative ELISA. The amount of HIV Env proteins in SHIV 89.6 VLP preparations was determined by a sandwich ELISA. ELISA plates were coated with a sheep-anti-gp120 antibody as the capture antibody, followed by addition of serial 2-fold dilutions of SHIV 89.6 VLPs lysed by 1% Triton X-100. The amount of HIV Env bound to the plate was then detected by sera from HIV-infected patients as the detecting antibody, followed by addition of HRP-conjugated Goat-antihuman antibody and development of color. Lysed SIVmac239 Gag VLPs were used as controls. A standard curve for the amount of HIV Env was obtained by adding purified HIV 89.6 Gp120 mixed with SIVmac239 Gag VLP lysed by 1% Triton X-100 to the sheep-anti-gp120 antibody-coated ELISA plate. The amount of HIV Env proteins was then calculated based on the obtained standard curve and then expressed as nanograms (ng) of HIV Env in 1  𝜇 g VLP preparation. VLP1, VLP2, and VLP3 represent SHIV 89.6 VLPs produced from three different batches and Gag-VLP represents the control SIVmac239 Gag only VLP preparation. (c) Negative staining and EM examination of SHIV 89.6 VLPs. Purified SHIV 89.6 VLPs were stained with 1% uranyl acetate followed by examination under a transmission electron microscope.