Extended Data Fig. 5: Engineering B cells with the sgRNA coded on the donor AAV.
From: In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice
a. Vector maps of the AAVs coding for the donorgRNA and the SFFV-Cas9. b. 3BNC117 IgG titers as quantified by ELISA over time in the SFFV-Cas9 + donorgRNA group. The black arrows indicate immunizations and the blue arrow indicates AAV injection. Each line represents a mouse. ***; pv = 0.0005 for Two-Way ANOVA comparing the SFFV-Cas9 + donorgRNA group to the donor group. n = 3 biologically independent mice. In this panel, the PBS and donor control groups are the same as for Fig. 2B. c. Transduction neutralization of TZM.bl cells by the YU2.DG (top) and JRFL (bottom) HIV pseudoviruses in the presence of day 136 sera IgGs. Neutralization is calculated as percent reduction from maximal luminescence per sample. The PBS control received immunizations, while the naïve control represents serum IgG from an untreated mouse. ns = non-significant, *; pv = 0.0462 and from top to bottom: ***; pv = 0.0007 and pv = 0.0004, ****; pv < 0.0001, Two-Way ANOVA with Šidák’s multiple comparison for time points comparison to PBS. d. Area under the Curve (AUC) for C. for YU2.DG and JRFL. ns; pv = 0.0667 (YU2.DG) and *; pv = 0.0103 (JRFL) for two-sided unpaired t-test for CMV-Cas9gRNA + donor to PBS comparison and ##; pv = 0.0097 (YU2.DG) and pv = 0.0078 (JRFL) for two-sided one-sample t-test for naïve to PBS comparison. n = 3 for CMV-Cas9gRNA + donor and PBS. Naïve sample from a single, non-immunized, non-AAV-injected mouse. In C-D, the PBS and control group is the same as for Fig. 2c and Extended Data Fig. 2a. Mean values ± SD are indicated. e. Representative flow cytometry analysis of 3BNC117+, CD19+, CD4− blood lymphocytes over time in the SFFV-Cas9 + donorgRNA group. f. Quantification of E. The black arrows indicate immunizations and the blue arrow indicates AAV injection. ###; pv = 0.0006 for Two-Way ANOVA comparison between groups and ****; pv < 0.0001 for Two-Way ANOVA with Šidák’s multiple comparison for time points comparison to PBS. For each group, each line represents the mean ±SD of n = 3 biologically independent mice. g. Representative flow cytometry analysis of 3BNC117+, CD19+, CD38+, CD138+ plasmablasts in the spleens of the SFFV-Cas9 + donorgRNA group at day 136. h. Quantification of G. Mean is indicated by the bars. ns; pv = 9892, ***; pv = 0.0005, one-way ANOVA with Tukey’s multiple comparison. i. Representative flow cytometry analysis of GL7+, Fas/CD95+ GC B cells in the spleens of the SFFV-Cas9 + donorgRNA group at day 136. j. Quantification of I. Mean is indicated by the bars. ns; pv = 0.8916, **; pv = 0.0075, one-way ANOVA with Tukey’s multiple comparison. k. Representative flow cytometry analysis of 3BNC117+ cells in total bone marrow (BM) of the SFFV-Cas9 + donorgRNA group at day 136. l. Quantification of 3BNC117+ CD19+ cells, ns; pv = 0.9965 and ****; pv < 0.0001 and m. Quantification of 3BNC117+ CD19− cells, ns; pv > 0.9999 and from top to bottom: ***; pv = 0.0003 and pv = 0.0003. Mean is indicated by the bars. One-way ANOVA with Tukey’s multiple comparison. n-o. Assessing overall immune homeostasis. Quantification by flow cytometry of total CD38+ CD138+ plasmablasts in spleen (N) ns; pv = 0.5622, total GL7+, Fas+ GC B cells in the spleen (O), at day 136 ns; pv = 0.9926. Mean is indicated by the bars.One-way ANOVA with Tukey’s multiple comparison. For E-O, the PBS and donor control groups are the same as for Fig. 3 and Extended Data Fig. 3.
