Fig 1.
Neutralization phenotype of parental and mutants of CH505TF produced in 293T and 293S GnT1- cells.
Neutralization tier phenotypes were characterized with pooled serum samples from 5 HIV-1 infected individuals (CHAVI samples). Additional neutralization phenotyping was performed with monoclonal antibodies. 1CH505TF.gly4 (197, 461/462, 276, 362); CH505TF.gly3.197 (461/462, 276, 362); CH505TF.gly3.276 (197, 461/462, 362); CH505TF.gly3.461 (197, 276, 362). 2GMT, geometric mean titer. 3Tier classification: Tier 1A (ID50 >2000), Tier 1B (ID50 350–2000), Tier 2 (ID50 50–349), Tier 3 (ID50 <50). ND, not done. Neutralization curves for the UCAs and intermediates of CH103 and CH235 are shown in S1A–S1K Fig.
Fig 2.
Neutralization by CH235 UCA2 predicts graded binding to corresponding SOSIP trimers.
(A) Overall strategy to identify and characterize engineered CH505TF Envs that engage CH235 UCA2. (B) Neutralization of parental and mutant CH505TF Env-pseudotyped viruses produced in either 293T or 293S GnTI- cells and assayed with CH235 UCA2 in TZM-bl cells. Shown are representative curves from S1H Fig. (C) SPR binding of CH235 UCA2 to parental and mutant CH505TF SOSIP trimers produced in either Freestyle293 (293F) or 293S GnT1- cells. The apparent dissociation rate constant (KD) values are an average of two measurements (see S3 Fig). NB, no binding.
Fig 3.
Cryo-EM structural details for reconstructions of CH235UCA complex with HIV-1 Env trimer CH505TF.N279K.G458Y.SOSIP.664.
(A) Cryo-EM reconstruction of CH235 UCA2 bound to stabilized CH505TF.N279K.G458Y.SOSIP.664/GnT1- trimer, shown segmented by component with CH235 UCA2 colored green and HIV-1 Env colored gray. (B) Zoomed-in view of the CH235 UCA2 binding interface with Env. The Env CD4 binding loop is shown in red, loop D in cyan, and loop V5 in orange. The CH235 UCA2 heavy chain is colored green, with the CHR H1, CDR H2 and CDR H3 loops colored purple, brown and blue, respectively. The CH235 UCA2 light chain is colored pink, with the CDR L1, CDR L2 and CDR L3 loops colored green, yellow, and magenta, respectively. The view on the right is rotated 90° clockwise with respect to the view shown on the left. (C) Zoomed-in view showing the interactions of the CH235 UCA2 CDR L3 (magenta) with the HIV-1 Env loop D (cyan) and loop V5 (orange). CDR H2 is shown in brown. The interactive residues are shown in stick representation and the black dotted lines indicate hydrogen bond interactions. (D) Left, cryo-EM reconstruction of the CH235 UCA2-Env complex shown in blue mesh with underlying fitted model shown in cartoon representation. Right, Zoomed-in view showing a quaternary interaction of the CH235 UCA2 heavy chain (green) N terminus with the adjacent protomer (wheat). Glycan 262 from the adjacent protomer is shown in stick representation.
Fig 4.
Immunization strategies to initiate and mature CH235 lineage bnAbs.
A strategy is presented to elicit CH235 lineage bnAbs by first priming with an immunogen that is predicted to engage appropriate naïve B cells with high, medium or low relative affinities. Subsequent boosting immunogens would aim to mature the response by driving the somatic mutations required to accommodate N279, G458 and a fully processed glycan shield. The choice of boosting immunogens depends in part on which priming immunogen proves to be optimal for precursor induction. Man5-enriched immunogens would be produced in GnT1- cells, and fully glycosylated immunogens would be produced in 293F or equivalent cells, where the cells for both immunogens are approved for GMP manufacturing.