Figure 1.

HIVCARs Based on bNAb Are Expressed on the Surface of Primary Human T Cells

(A) Known binding site for each bNAb scFv used indicated by color on a diagram of the HIV envelope. V1/V2, variable loops 1 and 2; mannose, high-mannose patch; CD4bs, CD4 binding site; MPER, membrane proximal external region. (B) Schematic diagram of the CAR construct in the pRRL LV backbone containing the γ-retrovirus-derived promoter-enhancer MND.⁶⁵ scFvs from various bNAbs (indicated by colored boxes below) were cloned upstream of the hinge region. CD8s, CD8-signaling domain; TM, CD8 trans-membrane domain; 4-1BB CD3z, intracellular signaling domains of second-generation CAR;⁶⁴ 2A, self-cleaving 2A peptide. (C) Percentage of BFP⁺ human primary CD3⁺ cells 5 days after LV transduction (tdx), and 8 days after enrichment by fluorescence-activated cell sorting (FACS). (D) MFI of BFP⁺ cells 8 days after enrichment. The bars in (C) and (D) show the mean ± SEM of n = 3 human cell donors. The same three donors were used for replicate transduction of each LV. (E) Representative flow plot showing surface CAR expression on primary human T cells transduced with pRRL MND VRC07-523-CAR T2A BFP.