Fig. 1. Generation of VRC01 gH mice and outline of priming experiments.

(A) Flow cytometry analysis of spleen cells showing B cell frequencies in VRC01 gH mice and WT littermates. (B) Next-generation sequencing of splenic cDNA from VRC01 gH mice revealed VH1-2*02 usage compared to mouse VH gene usage. (C) Summary of the time course for experiments and analysis. Mice were given a single prime of immunogen in adjuvant and then serum immune responses were evaluated at days 14, 28, and 42 post-immunization. Two of five mice per group were sacrificed for B cell sorting analysis at day 14, and the remaining three of five mice were sacrificed at day 42. In other animals, splenic B cells were collected at day 5, day 10, or day 31 for hybridoma generation. (D) Overview of the immunization groups listed by immunogen (eOD-GT8 60mer, eOD-GT8 3mer, eOD17 60mer, and BG505 SOSIP), multimeric state (nanoparticle or trimer), and adjuvant (Alum, Iscomatrix, and Ribi), along with the number of mice used to test each group. All groups were tested in both VRC01 gH and wild-type (WT) mice, except for BG505 SOSIP which was tested only in VRC01 gH mice. 5 mice per group were used for B cell sorting and/or ELISA, and an additional 13 VRC01 gH mice were employed for hybridoma generation after immunization with eOD-GT8 60mer (6 for Alum, 7 for Ribi).