Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

The sequence of iglb12 has been previously reported , and the CDRH3 and CDRL3 sequences are listed in Table S2 . Anti-idiotypic antibodies were generated by the Fred Hutchinson Cancer Research Center Antibody Technology Core. Briefly, 8-12-wk-old female BALB/c mice (Jackson Laboratory) were immunized and boosted intraperitoneally with iglb12 in adjuvant five times over a 5-mo period. 3 d following the final boost, splenocytes were electrofused to myeloma cells, and thousands of hybridoma colonies were screened for binding to iglb12 and a panel of control germline antibodies. Hybridomas producing antibodies specific for iglb12 were subcloned from single cells and expanded before anti-idiotype purification. Supernatant from these expanded hybridomas was harvested and purified over protein G resin (Pierce) following the manufacturer's recommendations. To produce recombinant antiidiotypes, RNA was extracted from 1 × 10 6 cells using the RNeasy kit (Qiagen) and the heavy and light chain sequences of the murine hybridomas were by obtained using the mouse Ig-primer set (69831; EMD Millipore) using the protocol developed by Seigel et al. (Siegel, 2009) . Sequences were codon optimized and cloned into pTT3-based IgG expression vectors with human constant regions (Snijder et al., 2018) using In-Fusion cloning (Clontech).