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.

To generate anti-idiotype antibodies to iglb12 , we immunized and boosted mice intraperitoneally with iglb12 in adjuvant. 3 d after the final immunization, splenocytes were isolated and fused to myeloma cells, resulting in the generation of hundreds of hybridomas producing antibodies able to bind iglb12. Nearly all of the clones were excluded because they bound to a panel of control germline antibodies as well as iglb12, suggesting specificity for the heavy or light chain constant regions of human Ig (data not shown). Importantly, four clones (IB1, IB2, IB3, and IB5) exhibited binding to iglb12 with similar apparent affinities (dissociation constants [K D ] of ∼10 −9 ) but did not bind a panel of control antibodies derived from germline BCR sequences of anti-HIV-1 antibodies (Fig. 1 , A-C; and Table S1 ). The anti-iglb12 idiotypes also failed to bind the fully mature version of b12, indicating a preference for the unmutated sequence ( Fig. 1 C) . One clone, IB2, bound a chimera expressing the iglb12 heavy chain and a noncognate control light chain, while the other three clones, IB1, IB3, and IB5, did not bind this chimera ( Fig. 1 C) , suggesting different modes of recognition between IB2 and IB1, IB3, and IB5. We next assessed whether binding of these clones was specific for the iglb12 heavy chain CDRH3 region as opposed to a dependence on V H 1-3, the variable gene segment used by iglb12. Consistent with our clones specifically recognizing features unique to iglb12, all four clones failed to bind naturally paired control antibodies using V H 1-3 (DeKosky et al., 2016) whether or not they were paired with a V k 3-20 light chain, from which the b12 light chain is derived ( Fig. 1 C) . Together, these data suggest that these four clones preferentially recognized the iglb12 idiotype.