3A and 3D). maturation is necessary for cross-reactivity to diverse isolates: bnAb inferred-germline variants typically show detectable affinity for few if any HIV envelope protein (Env) antigens tested (35,7,1021). Most wild-type Env proteins are therefore poor immunogens to prime bnAb responses (35,17,22,23). Reliable vaccine initiation of bnAb responses will likely require design or discovery of germline-targeting priming immunogens with appreciable affinity for bnAb germline precursors (35,10,11,17,2229). Furthermore, consistent bnAb priming may only be possible for bnAb precursors present at reasonable frequency in all or PF-05180999 most vaccine recipients (27). Proof of principle that a germline-targeting immunogen can prime relatively rare bnAb-precursor B cells was shown with the eOD-GT8 60 subunit self-assembling nanoparticle (60mer) that targets precursors of CD4-binding-site-directed VRC01-class bnAbs (26). VRC01-class precursors are defined by their use of a heavy chain VH12*02 (or *03 or *04) gene and light chains with unusually short complementarity determining region 3 (CDR3) loops of five amino acids (5,3032). In a transgenic mouse model expressing the germline-reverted VRC01 heavy chain paired with wild-type mouse light chains (VRC01 gH mouse), in which the frequency of VRC01-class precursor B cells was estimated to be higher than in humans by a factor of as little as ~5, a single immunization with eOD-GT8 60mer activated VRC01-class precursors and generated VRC01-class memory responses in nearly all immunized mice, while control immunogens presenting a native CD4 binding site failed to activate PF-05180999 such precursors (26). The eOD-GT8 60mer activated target precursors less robustly in a different VRC01-class inferred-germline heavy chain transgenic mouse (25). Several properties of both mouse models lowered the bar for germline-targeting compared to the challenges confronted in a human: elevated bnAb precursor frequency, reduced competition from other B cell specificities, limited diversity of bnAb precursors owing to their uniform recombined heavy chains, and an affinity advantage conferred on bnAb precursors due to their PF-05180999 mature H-CDR3s. To better model the conditions for initiation of a VRC01-class bnAb response in humans, we investigated immunization with eOD-GT8 60mer in Kymab mice transgenic for the complete un-rearranged human antibody germline gene repertoire (33). Kymab mice contain human heavy chains paired with either human kappa light chains (HK mice) or human lambda light chains (HL mice), or both (HKL mice). To determine the level of difficulty for VRC01-class bnAb priming in Kymab mice, we first measured the frequency of VRC01-class precursors in the above mouse strains. Using B cell sorting methods, we previously detected eOD-GT8-specific VRC01-class precursors at a frequency of 1 1 in 2.4 million among human nave B cells expressing kappa light chains (27). Using similar methods to probe a total of approximately 300 million nave B cells from the spleens and lymph nodes of unimmunized HK and HL mice (N=3 each), we were unable to isolate any VRC01-class B cells, suggesting that SPN the frequency of such B cells was considerably lower than in humans. The frequency of the VH12*02, *03, or *04 alleles among HK B cells was previously measured as 0.9% (33,34), lower than the frequency in humans (2.91.3%) (fig. S1) (35,36) by a factor of only ~3. Therefore, to explain the reduced frequency of VRC01-class B cells in Kymab mice, we analyzed the light chain variable gene (VL) usage and CDR3 length distributions from IgM+ or IgG+ B cells for five HK mice and four healthy humans by next-generation sequencing (NGS) (37). The kappa light chain-CDR3 (L-CDR3) length distributions were broadly similar in HK mice and humans (Fig. 1A), but the frequency of 5-amino acid L-CDR3s was lower in HK mice (0.018 0.013%) than in humans (0.95 0.57%) by a factor of approximately 50 (Fig. 1B). The cumulative frequency of V genes used by known VRC01-class bnAbs (IGVK320, IGVK133 and IGVK315 (32,38)) was 24.0 3.5% in HK mice, similar to the 23.2 4.9% that we measured in humans (Fig. 1C). However, the frequency of 5-amino acid L-CDR3s associated with known VRC01-class kappa chains was reduced by a factor of approximately 300 in HK.
