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.

Env-based immunogens capable of binding to certain germline bNAbs have been recently developed that effectively activate B cells in knock-in mice expressing the desired BCRs in vivo (Dosenovic et al., 2015; Escolano et al., 2016; Jardine et al., 2016; McGuire et al., 2016; Steichen et al., 2016; Abbott et al., 2018) . For many bNAbs, however, antigens capable of being bound by the corresponding germline forms have not yet been identified or designed. Here, we demonstrate the use of anti-idiotypes as an alternative approach to structure-based immunogen design to target the inferred germline version of the HIV-1 bNAb b12. We focused upon b12 because despite being one of the first HIV-1 bNAbs isolated, Env-based immunogens have not been identified or developed to recognize its ancestral germline form (Xiao et al., 2009; Hoot et al., 2013) . The mature form of b12 was isolated through phage display (Barbas et al., 1992) , and it used a heavy chain derived from V H 1-3*02/D H 2-21/J H 6*03 and light chain derived from V K 3-20*01/J K 2 (Xiao et al., 2009; Hoot et al., 2013) . Using anti-idiotypes specific for the inferred germline version of b12 (iglb12) as baits for single B cell sorting, we identified a subset of human germline BCRs using V H 1-3 with some heavy chain CDRH3 similarity to iglb12. While crystal structures indicated that one of these anti-iglb12 idiotypes made extensive contacts with the iglb12 CDRH3 region encoded by D H 2-21, this gene segment was not enriched in sorted B cells. Moreover, we could not identify any CDRH3 regions with >50% amino acid sequence identity to iglb12 within a dataset including hundreds of BCRs. In light of this, we hypothesized that B cells expressing BCRs containing CDRH3s with high similarity to iglb12 may be deleted from the repertoire as a result of autoreactivity with self-antigens. In line with this notion, we demonstrate that the iglb12 antibody stains HEp-2 cells, a feature of other autoreactive HIV-1-specific antibodies (Haynes et al., 2005; Verkoczy et al., 2010; Ota et al., 2013) . To evaluate the consequence of this autoreactivity in vivo, we generated mice with the iglb12 heavy chain knocked into the endogenous murine heavy chain locus. The iglb12 heavy chain knock-in mice exhibited B cell deletion during development, with the surviving B cells exhibiting BCR down-regulation and anergy. Despite the presence of anergy, immunization with a multimerized version of an anti-iglb12 idiotype stimulated the proliferation and differentiation of transgenic B cells expressing the iglb12 heavy chain. In summary, our results establish a proof of concept that anti-idiotypes can be used as immunogens to identify and stimulate the ancestral germline version of protective antibodies.