Only a few passages of library viruses in the presence of a CD4bs bNAb is sufficient for escape viruses to emerge.

(A) Neutralization assays of the passage 0–5 swarms selected against NIH45-46. Assays were performed in TZM-bl reporter cells. ‘Parental library’ is the virus library without any passage, and ‘Control passage 5’ is the parental virus library passaged 5 times in the absence of NIH45-46. (B) Neutralization assays with the NIH45-46 passage 5 swarm against the indicated CD4bs bNAbs. VRC01 and VRC07 were isolated from the same donor as NIH45-46 (donor: NIH45), but 3BNC117, VRC-PG04, VRC-CH31 and VRC-PG20 were isolated from Patient 3, Donor 74, CH0219 and IAVI 23, respectively. Averages ± SD of three independent experiments performed in duplicates are shown. Statistical significance was calculated by two-way ANOVA and p value was <0.001 for all differences with the relevant control swarm.