A 4?L aliquot of the sample was applied onto a glow-discharged C-Flat 1.2/1.3 grid (Electron Microscopy Sciences, Protochips, Inc.), blotted and then plunged into liquid ethane using FEI Vitrobot Mark IV to capture complexes in vitreous snow. address these limitations, we devised a vesicular stomatitis computer virus (VSV)-centered probe to display membrane-embedded Env trimers and isolated five bNAbs from two chronically infected donors, M4008 and M1214. Donor B cell receptor (BCR) repertoires recognized two bNAb lineages, M4008_N1 and M1214_N1, that class-switched to immunoglobulin G (IgG) and IgA. Variants of these bNAbs reconstituted as IgA shown broadly neutralizing activity, and the IgA portion of M1214 plasma conferred neutralization. M4008_N1 epitope mapping exposed a glycan-independent V3 epitope conferring tier 2 computer virus neutralization. A 4.86-?-resolution cryogenic electron microscopy (cryo-EM) SMAD9 structure of M1214_N1 complexed with CH505 SOSIP revealed another elongated epitope, the V2V5 corridor, extending from V2 to V5. Overall, the VSVENV probe recognized bNAb lineages with neutralizing IgG and IgA users targeting unique sites of HIV-1 Env vulnerability. Construct and Probe Preparation As explained (Liberatore et?al., 2019), the plasmids encoding full-length VSV genome (pVSV-FL) as well as individual VSV genes N, P, L, and G were purchased from Kerafast (Boston, MA). The plasmid encoding the HIV-1 strain AD17 was from the NIH AIDS Reagent System, as contributed by Drs. Beatrice Hahn and George Shaw. The chimeric Env was generated using overlap-extension PCR, in which the extracellular and transmembrane domains of AD17 Env were fused to the cytoplasmic tail of VSV-G. The chimeric Env DNA fragment was put into pVSV-FL exactly in place of the VSV-G MK 0893 to generate pVSVAD17. A plasmid bearing VSV cDNA encoding a monomeric NeonGreen-phosphoprotein P fusion protein (mNG-P) has been explained (Kleinfelter et?al., 2015, Spence et?al., 2016). Here, the VSV-G in pVSV-mNG-P was replaced with the chimeric Env DNA fragment to generate pVSVAD17-mNG-P. The VSVAD17 computer virus was rescued by infecting 293T cells with T7-expressing vaccinia (vTF7-3) at a MOI of 5, followed by transfection with pVSVAD17 (or pVSVAD17-mNG-P) and plasmids encoding VSV-N, P, L, and G under the control of a T7 promotor. Supernatant MK 0893 was harvested in 48 h, slowly filtered (0.22?m) to remove vaccinia and plaque purified on GHOST.R5 cells. Plaque purified VSVAD17 was expanded in GHOST.R5 cells, filtered (0.22?m), and stored in aliquots at ?80C. To prepare VSVAD17-PE, an aliquot of VSVAD17 was used to infect a T-75 flask of GHOST.R5 cells; at 24?h after illness, 20?mL of freshly collected VSVAD17 supernatant was filtered (0.22?m) and pelleted by ultracentrifugation; the MK 0893 viral pellet was then labeled with PE (Abcam, Cambridge, MA). To prepare VSVAD17-mNG, an aliquot of VSVAD17-mNG was used to infect a T-75 flask of pVSV-G transfected GHOST.R5 cells; at 24?h after illness, the VSVAD17-mNG (decorated with VSV-G) supernatant MK 0893 was MK 0893 filtered (0.22?m) and used to infect (single-round illness) a T-75 flask of pre-seeded 293T-furin cells; at 24?h after illness, the 293T-furin cells were inspected under green fluorescent microscope for 90% confluence and 90% green fluorescence; 20?mL of freshly collected VSVAD17-mNG supernatant was filtered (0.22?m) and directly used to stain B cells. Fluorescence Activated Cell Sorting, Solitary B cell RT-PCR, Antibody Manifestation and Purification Patient or blood donor PBMC samples were pre-sorted for B cells by FACS. Briefly, donor PBMCs were stained with anti-human CD3-PE-CF594 (BD Biosciences, San Jose, CA), CD19-PE-Cy7 (BioLegend, San Diego, CA), and CD20-APC-Cy7 (BioLegend), and the CD3-CD19+ B cells were bulk sorted. In addition, live/lifeless yellow stain (Invitrogen) was used to exclude lifeless cells. The bulk sorted B cells were then re-stained with anti-human CD3-PE-CF594, CD19-PE-Cy7, CD20-APC-Cy7, with additional staining of IgM-V450 (BD Biosciences), IgG-FITC (BD Biosciences) and VSVAD17-PE, or IgM-V450 and CD27-PerCP-Cy5.5 (BD Biosciences) with VSVAD17-mNG. Fluorescence payment was performed with anti-mouse Ig beads (BD Biosciences) stained with each antibody in a separate tube. After washing, cells were analyzed and sorted using a multi-laser MoFlo sorter (Beckman Coulter, Jersey City,.