Fig 1.
Boosting of RV144 vaccinees increased VH chain gene mutation frequency and caused a repertoire shift, increasing the frequency of antibodies with Heavy Chain Complementary Determinant Region 3 (HCDR3) ≥ 22 amino acids.
PBMCs from four vaccinees post-RV144 (A&B) and post-RV305 (C&D) were antigen-specific single-cell sorted with fluorophore labeled conjugates. The VH/VL chain genes were PCR-amplified and screened for Env-reactivity by ELISA. The VH chain gene mutation frequency and HCDR3 lengths of 145 Env-reactive antibodies from RV135/144 and 242 Env-reactive antibodies from RV305 were analyzed with Cloanalyst[56]. (A&C) Error bars represent the standard error of the mean.
Fig 2.
The long Heavy Chain Determinant Region 3 (HCDR3) antibodies that neutralize virus bind the CD4 binding site (CD4 bs).
Purified recombinant monoclonal antibodies (mAbs) were assayed by ELISA for (A) blocking the binding of soluble CD4 to AE.A244gp120, and (B) sensitivity to the CD4 binding site mutations Δ371I/P363N, D368R, N276A, and T278A in the AE.A244gp120 protein. (C) Epitope mapping of mAbs on yeast displayed YU2gp120 with point mutations within the inner domain, outer domain and CD4 binding site. (D) Assaying the long HCDR3 CD4 bs antibodies for HIV-1 neutralization in the TZM-bl neutralization assay.
Fig 3.
Neutralization of the tier 1 HIV-1 isolate AE.92TH023 and the tier 2 HIV-1 isolate CRF_01 AE.CNE8 by the DH576 Unmutated Common Ancestor (UCA) and DH576 lineage members.
The DH576 clonal lineage was inferred with Cloanalyst[56]. The DH576 clonal lineage was assayed for neutralization of the tier 1 isolate AE.92TH023 and the tier 2 isolate CRF_01 AE.CNE8 in the TZM-bl neutralization assay. Affinity maturation of the DH576 clonal lineage improved neutralization for the tier 1 isolate AE.92TH023 by > 3 logs and for the tier 2 isolate CRF_01 AE.CNE8 by approximately 1 log.
Fig 4.
Negative stained electron microscopy of DH576 in complex with CH505 SOSIP.664.
(A) 2D class averages of one, two, or three DH576 Fabs bound to the trimer and (B) Side and top views of the 3D EM reconstruction with the indicated Ab or sCD4 superimposed on the gp120 subunit for comparison with DH576 binding. Arrows indicate the angle at which the indicated antibody approaches the trimer.