BG505 3mut-DS-SOSIP.664 shows enhanced antigenic specificity, thermostability, and immunogenic specificity. (A) Antigenicity, as determined by MSD, of stabilized BG505 DS-SOSIP.664 trimers after V3-negative selection assessed on a panel of antibodies, including nonneutralizing/weakly neutralizing CD4-induced antibodies (17b and 48d with and without soluble CD4, and F105), CD4-binding site antibodies (VRC01, VRC13, and b12), broadly neutralizing V1/V2 antibodies (PGT145, CAP256-VRC26.25), broadly neutralizing glycan-V3 antibodies (PGT121, PGT128, and 2G12), nonneutralizing/weakly neutralizing V3-directed antibodies (447-52D, 3074, and 2557, with and without soluble CD4), broadly neutralizing gp41-gp120 interface antibodies (35O22 and 8ANC195), and fusion peptide antibody (VRC34.01 and PGT151). (B) Thermostability of stabilized BG505 Env trimers assessed by DSC. Top, raw data from DSC shown in solid lines, with A329P-DS-SOSIP.664 shown in orange, 2mut-DS-SOSIP.664 shown in blue, 3mut-DS-SOSIP.664 shown in magenta, 5mut-DS-SOSIP.664 shown in cyan, 4mut-DS-SOSIP.664 shown in green, DS-SOSIP.664 shown in red, and BG505 SOSIP.664 shown in black. Bottom, Tm, ΔT1/2 (width at half-peak height), and enthalpy of unfolding from DSC (ΔH). (C) Binding parameters of stabilized HIV-1 Env trimers to the soluble CD4 D1D2 domain. Values in parentheses report standard errors from fitting data to a 1:1 Langmuir binding model. (D) Autologous neutralization titer (BG505, left) and MW965.26 neutralization titer (right) of guinea pig sera at week 18 (after three trimer injections). Statistical differences between BG505 3mut-DS-SOSIP.664 and BG505 SOSIP.664 or BG505 DS-SOSIP.664 were calculated using a one-tailed Mann-Whitney test. AUC, area under the curve; Cp, heat capacity; KD, equilibrium dissociation constant; ka, absorption rate constant; kd, dissociation constant.