The kinetics of trimer and nanoparticle binding to bNAbs, non-NAbs, and CD4-Ig was measured using an Octet Red96 instrument (fortéBio, Pall Life Sciences). All assays were performed with agitation set to 1000 rpm in fortéBio 1× kinetic buffer. The final volume for all the solutions was 200 μl per well. Assays were performed at 30°C in solid black 96-well plates (Geiger Bio-One). Antibody (5 μg ml−1) in 1× kinetic buffer was loaded onto the surface of anti-human Fc capture (AHC) biosensors for 300 s. A 60-s biosensor baseline step was applied before the analysis of the association of the antibody on the biosensor to the antigen in solution for 200 s. A twofold concentration gradient of antigen, starting at 200 nM for trimers and 14 to 35 nM for nanoparticles depending on the size, was used in a titration series of six. The dissociation of the interaction was followed for 300 s. Correction of baseline drift was performed by subtracting the mean value of shifts recorded for a sensor loaded with antibody but not incubated with antigen and for a sensor without antibody but incubated with antigen. Octet data were processed by fortéBio’s data acquisition software version 8.1. Notably, for apex-directed bNAbs, experimental data were fitted with the binding equations describing a 2:1 interaction to achieve the optimal fitting results. CD4-Ig binding experiments were performed following the same procedure described above. Paired t test in Prism (two groups, n = 10) was used to determine whether trimer-antibody binding signals were statistically different (P < 0.05) upon gp41ECTO swapping.

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