The antibodies did not compete with a previously isolated glycan holeCspecific antibody but did compete with N332 glycan supersite broadly neutralizing antibodies. broadly neutralizing responses to the N332 glycan supersite and vaccine immunogens may require engineering to minimize these off-target responses or steer them toward a more desirable pathway. INTRODUCTION Given their protective efficacy in passive transfer studies, the elicitation of broadly neutralizing antibodies (bnAbs) is one of the primary objectives of current HIV research ((National Research Council, 1996). Rabbit 5743 serum exhibited Schisandrin A strong autologous neutralization of the BG505 pseudovirus, but unlike neutralizing sera of most rabbits, 5743 also neutralized the closely related MG505.A2 strain, where a lysine at position 241 abrogates GH neutralization. Thus, the objective of this study was to characterize and map the response(s) exhibited by rabbit 5743 serum. Toward that end, we isolated PBMCs and performed BG505-specific B cell sorting to isolate the relevant mAbs. Subsequently, site-directed mutagenesis of pseudoviruses and ELISAs were used to approximate the BG505 epitope to which the 5743 mAbs were elicited, and whether Rabbit Polyclonal to CYSLTR1 these isolated mAbs overlap with known bnAbs. To determine the degree to which glycans were involved in the paratope-epitope interactions, we also performed neutralization assays with deglycosylated BG505 and MG505.A2 pseudoviruses. To visually confirm the epitope of the 43A class of mAbs, negative-stain EM was used, followed by high-resolution cryoEM with 43A2 to elucidate the details of the BG505 epitopeC43A2 paratope toward informing immunogen design. Isolation of rabbit B cells Cryopreserved PBMCs from rabbit 5743 were thawed, resuspended in 10 ml of RPMI 10% fetal calf serum (FCS), and collected by centrifugation at 600for 5 min. Cells were washed with phosphate-buffered saline (PBS), resuspended in 10 ml of PBS, and collected by a second centrifugation step. Cells were resuspended in 100 l of FWB (2% FCSCPBS) with anti-rabbit immunoglobulin M (IgM) fluorescein isothiocyanate (FITC) (1:1000) and a streptavidin-allophycocyanin (APC) tetramer of biotinylated anti-rabbit IgG. After 1 hour on ice, cells were washed once with 10 ml of PBS, collected by centrifugation, and resuspended in 100 l of FWB with 1 l of a streptavidin-phycoerythrin (PE) tetramer of biotinylated BG505 or B41 SOSIP.664. After a further 1 hour on ice, cells were washed once with 10 ml of PBS, collected by centrifugation, and resuspended in 500 l of FWB for sorting on BD FACSAria III. IgM?IgG+BG505+B41 lymphocytes were collected at one cell per well into SuperScript III Reverse Transcriptase lysis buffer (Invitrogen) as previously described and immediately stored at ?80C before complementary DNA generation and single-cell polymerase chain reaction. Generation of antibodies and Fabs Rabbit antibody variable regions (GenBank accession number: KX571250-1324) were cloned into an expression plasmid adapted from your pFUSE-rIgG-Fc and pFUSE2-CLIg-rK2 vectors (InvivoGen). Human and rabbit antibodies were transiently expressed with the FreeStyle 293 Expression System (Invitrogen). Antibodies were purified using affinity chromatography Schisandrin A (Protein A Sepharose Fast Flow, GE Healthcare), and the purity and integrity were checked by SDSCpolyacrylamide gel electrophoresis. To generate Fabs, rabbit IgG was digested with 2% papain (Sigma, P3125) in digestion buffer [10 mM l-cysteine, 100 mM Na acetate (pH 5.6), 0.3 mM EDTA] for 6 hours and then quenched with 30 mM iodoacetamide. Undigested IgG and Fc fragments were removed by affinity chromatography, and the Fab-containing circulation through was collected. Size-exclusion chromatography was performed using Superdex 200 10/300 resin (GE Healthcare) to remove papain and digestion by-products. Neutralization assays Pseudovirus neutralization assays using TZM-bl target cells were carried out as previously explained (clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 79, 10108C10125 (2005). [PMC free article] [PubMed] [Google Scholar] 39. Seaman M. S., Janes H., Hawkins N., Grandpre L. E., Devoy C., Giri A., Coffey R. T., Harris Schisandrin A L., Solid wood B., Daniels M. G., Bhattacharya T., Lapedes A., Polonis V. R., McCutchan F. E., Gilbert P. B., Self S. G., Korber B. T., Montefiori D. C., Mascola J. R., Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J. Virol. 84, 1439C1452 (2010). [PMC free article] [PubMed] [Google Scholar] 40. Sanders R. W., van Gils M. J., Derking R., Sok D., Ketas T. J., Burger J. A., Ozorowski G., Cupo A., Simonich C., Goo L., Arendt H., Kim H. J., Lee J. H., Pugach P., Williams M., Debnath G., Moldt B., van Breemen M. J., Isik G., Medina-Ramrez M., Back J. W., Koff W. C., Julien J.-P., Rakasz E. G., Seaman M. S., Guttman M., Lee K. K., Schisandrin A Klasse P. J., LaBranche C., Schief W..
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