Fig 1.
Purification of the full-length AMC011 trimer in complex with PGT151 or PGT145 Fab.
(A) Size exclusion chromatography profiles of full-length AMC011 Env bound to PGT151 Fab (two runs) or bound to PGT145 Fab (one run). (B) Average yields of full-length Env constructs bound to PGT151 Fab or PGT145 Fab and their SOSIP.664 counterparts. For comparison, soluble AMC011 SOSIP.664 trimers were purified with PGT151 and PGT145 affinity chromatography columns. The mean yields shown in the graph represent the averages of three to six purifications. (C) Thermostability of full-length AMC011 trimers in DDM or at different concentrations of DOPC and DOPC:CHS (1:1 molar ratio) after detergent removal. The unfolding pattern of the different proteins was assessed by plotting the first derivative of the curves acquired with nano-DSF. The Tm onset, the melting temperatures of the Env (Tm Env) and the Tm of the antibody (Tm Ab) are shown in the table. The averages Tm values derived from two to five different experiments are listed in the table. The raw data of one such experiment is depicted in the graph. NA: not applicable. (D) 2D class averages from negative-stain EM images are shown for the full-length AMC011 Env trimer bound to PGT151 Fab in DDM or at different concentrations of DOPC or DOPC:CHS (1:1 molar ratio). Colors in the thermostability graph match the 2D class average boxes. (E) Thermostability of full-length AMC011 Env trimer bound to PGT145 Fab in 0.1 mM DOPC:CHS (1:1 molar ratio). The Tm was calculated from one experiment. (F) Representative 2D class averages from full-length AMC011 Env trimer in 0.1 mM DOPC:CHS. (G) 3D models of full-length AM0C11 trimer bound to PGT151 or to PGT145, which were derived from the class-averages shown in (D) and (F). Coloring in (D), (F) and (G) is as follows: PGT151 and PGT145 in light orange, the bicelle in gray and the envelope trimer in dark blue.
Fig 2.
Antigenicity profile of purified full-length AMC011 Env trimer determined by BLI.
(A) The antigenicity of full-length and SOSIP.664 Env trimers was assessed by BLI (Octet). Bars represent the maximum values of the binding curves (S2 Fig). A panel of 16 bNAbs and 5 non-NAbs was tested: apex directed antibodies (green), V3-glycan (blue), CD4 binding site (black), gp120-gp41 interface (magenta), MPER (brown) and non-NAbs (red). ND: not determined (the lower yields of full-length AMC011 Env in complex with PGT145 precluded a full analysis). The values are representative of two independent experiments. The curves from one representative experiment are shown in S2 Fig. (B) The binding of bNAbs and non-NAbs to full-length and to SOSIP.664 Env trimers, assessed by BLI, was compared. The Spearman correlation (inset) is shown. For BLI values in which both constructs report data, the average of both was used. (C) Same analysis as in (B), but excluding the non-NAbs and MPER-directed NAb. Antibodies are colored as in panel (A).
Fig 3.
Antigenicity profile of the full-length Env protein present on the cell membrane or on the virus.
(A) Neutralization of the AMC011 virus was assessed as a proxy for antibody binding to the functional AMC011 trimer on the virus. IC50 and maximum percentage neutralization (MPN) values for a panel of bNAbs and non-NAbs against AMC011 virus are shown. Binding of bNAbs and non-NAbs to the Env protein embedded in the membrane of HEK293F cells was determined by FACS. Half maximal binding concentrations (EC50) and area under the curve (AUC) values are shown. Data are representative for two to four different experiments. (B) Comparison of AMC011 antigenicity obtained by FACS (membrane-bound Env) and neutralization assays (virus-associated functional Env), by FACS and BLI (purified membrane-derived Env) (C) and by neutralization assays and BLI (D) are shown.
Fig 4.
Cryo-EM structure of full-length AMC011 Env in complex with PGT145 Fab or PGT151 Fab and comparison with other structures.
(A) The cryo-EM map of the full-length AMC011 trimer bound to PGT151 Fab (left), the reconstruction of which is at 4.2 Å and the cryo-EM map of the full-length AMC011 trimer bound to PGT145 Fab (right), the reconstruction of which is at 5.7 Å are shown. Superimposition of the full-length AMC011 Env bound to PGT145 Fab (in green) with: (B) full-length AMC011 bound to PGT151 Fab (in gray) and (C) AMC011 SOSIP.v4.2 (in light red). Superimposition of the full-length AMC011 Env bound to PGT151 Fab (in gray) with: (D) AMC011 SOSIP.v4.2 (in light red).
Fig 5.
Binding of PGT145 Fab or PGT151 Fab to full-length AMC011 trimer.
(A) Superimposition of the cryo-EM map of the full-length AMC011 Env bound to PGT151 Fab (blue) and the JR-FL ΔCT trimer bound to PGT151 Fab low-pass filtered at 4.2 Å (gray, Lee et al., 2016). (B) Close-up of the PGT151 epitope. Coloring is as follows: glycans are depicted in green, PGT151 Fab in blue and the cryo-EM reconstruction density in white. (C) Superimposition of the cryo-EM map of and AMC011 full-length trimer bound to PGT145 and BG505 SOSIP.664 bound to PGT145 low-pass filtered to 5.7 Å. The vertical and horizontal axes were used to compare tilt and rotation angle between both PGT145 Fabs. (D) Close-up of the PGT145 epitope. The crystal structure of the PGT145 Fab (PDB: 3U1S) was docked into both cryo-EM maps for epitope comparison. The cryo-EM map of full-length AMC011 trimer bound to PGT145 is shown in white. (E) Cross sections of the PGT145 epitope (box in (D)) in BG505 SOSIP (gray) and AMC011 full-length (blue) trimers. Residue Pro124 of the trimer is shown in stick as a direct contact with the Fab. (F) Distribution of N160 glycans in the PT145 epitope of BG505 SOSIP and AMC011 full-length trimer. PGT145 Fab crystal structure (dark gray and dark blue for BG505 and AMC011, respectively) was docked into cryo-EM maps of BG505 SOSIP (EMD-8644) and AMC011 full-length trimers. The first sugars of the N160 glycan are indicated in sticks.
Fig 6.
Glycan composition of full-length and SOSIP.664 AMC011 trimer.
(A) Comparison of N-linked glycan profiles of full-length AMC011 trimer and SOSIP.664 Env trimer. Full-length AMC011 Env was purified in complex with PGT151 Fab. AMC011 SOSIP.664 was purified by PGT151 affinity chromatography. Green indicates oligomannose-type and hybrid glycans. Complex glycans are shown in magenta. (B) Relative quantification of distinct glycan types of full-length AMC011 and AMC011 SOSIP.664. The bar graphs show the relative percentage of each glycoforms at a particular site and the pie charts summarize the amount of oligomannose-type (green) and complex/hybrid (magenta) glycans on each glycan site. N.A.: not assessed. (C) Glycan sites are colored according to their processing state in the AMC011 SOSIP.v4.2 cryo-EM map low-pass filtered to 30 Å: in green, oligomannose-type; in magenta, processed glycans; in orange, mixed glycans; in gray, glycans that could not be assigned.