Fig 2: Effects of IP or IV administration of aCD20 on splenocyte B cell populations in C57Bl/6 mice. Splenocyte B cell (anti-CD19 antibody) and T cell (anti-CD3 antibody) populations were quantified using flow cytometry 7 days after the administration of the aCD20 antibody (SA271G2 BioLegend). (A–C) IV administration of either 50µg or 100µg aCD20, showing representative flow cytometry plots (A), total cell numbers (B), and cell population proportions (C). (D, E) IP administration of either 25 µg or 100 µg aCD20, showing representative flow cytometry plots (D), total cell numbers (E), and cell population proportions (F). (G) Effects of B cell depletion on B cell subsets for IP treated mice; marginal zone (CD5-CD21+CD23-IgM+), follicular (CD5-CD23+IgM-) and regulatory (CD5+CD21+IgM+) B cell populations are shown. For all histograms columns represent means, error bars SDs: black bars, undepleted; gray bars, low-dose depleted; white, high-dose depleted (n=3). Mann Whitney U test was used for statistical analysis (***P < 0.001, ****P < 0.0001).

Fig 3: Chromatin state of the DH-CH domain of the IgH locus in CD4+CD8+ DP thymocytes. (A) Top panel shows scale representation of the murine IgH locus based on mm9 (mouse reference genome). VH gene families are indicated in blue. Purple box labeled as DJ contains all DH and JH gene segments, and pink box labeled as C contains exons of all IgH isotypes. The DH-CH domain (120 kb) is expanded below to show the locations of ChIP amplicons (black bars). The intergenic control region 1 (IGCR1), DQ52 promoter (PQ52), and intronic enhancer (Eµ) are regulatory sequences that are marked by DNase I hypersensitive sites in pro-B cells. (B) CD19+ pro-B cells from bone marrow of Rag2-/- and CD4+CD8+ thymocytes from TCRß × Rag2-/- transgenic mice were used in chromatin immunoprecipitation (ChIP) experiments using anti-H3K4me3 and H3K9ac antibodies. TCF7 and Lck gene promoters served as positive controls in DP thymocytes. C?3 was used as a negative control and ?-actin served as a positive control in both cell types. For each independent experiment PCR was done in triplicate. Results shown are the mean of two independent experiments. Error bars represent standard error of the mean (n = 2). Y-axis shows enrichment of respective amplicons in the immunoprecipitate compared to an equal amount of input DNA as described in the methods. (C) DNase I sensitivity analysis of proximal part of IgH locus in pro-B cells and DP thymocytes. 107 nuclei from CD19+ pro-B cells from Rag2-/- mice and DP thymocytes from TCRß × Rag2-/- mice were treated with increasing concentration of DNase I (X-axis) followed by purification of genomic DNA. Equal amounts of DNA were used for amplification with the indicated primers. The signal from each amplicon was normalized to that from a ß-globin amplicon for each DNase I concentration. ß2m promoter served as a positive control while C?3 served as negative control. TCRa enhancer was used as an additional positive control for DP thymocytes. The data represents the mean of two independent experiments. Error bars represent standard error of the mean (n = 2).

Fig 4: Immunogenicity of GAd-ZIKV Vaccines in WT C57BL/6 Mice(A) Schematic representation of the transgene cassettes. GAd-control has no transgene insert, whereas GAd-Eecto and GAd-Zvp contain ZIKV (strain H/PF/2013) a full prM-E cassette or prM-E ectodomain, respectively.(B) A549 cells were infected with GAd-Eecto or GAd-Zvp followed by concentration of the supernatants by ultracentrifugation, negative staining, and transmission electron microscopy imaging to confirm the production of VLPs. High-power images are shown and compared to ZIKV VLP preparation generated by transfection of prM-E genes. Scale bar, 100 nm.(C) ZIKV E protein expression in GAd-Eecto or GAd-Zvp. A549 cells were infected with GAd-Eecto, GAd-Zvp, or mock-infected, and supernatants were prepared and run on SDS-PAGE under reducing (upper blot) or non-reducing (lower blot) conditions along with recombinant ZIKV E ectodomain protein followed by detection using ZV-48. One representative blot from three experiments is shown.(D-H) C57BL/6 mice were immunized with 109 viral particles of GAd-control, GAd-Eecto, or GAd-Zvp via intramuscular inoculation and boosted 3 weeks later.(D and E) Humoral responses were evaluated in the sera of immunized mice at day 21 post-prime and day 21 post-boost. An ELISA measured ZIKV E-specific binding IgG (D), and a FRNT determined neutralization activity (EC50 values are shown) (E). Data for humoral responses are pooled from two experiments with n = 10–18 per group (ANOVA with a Dunnett’s post-test comparing vaccine and control groups: ****p < 0.0001).(F) Localization of GAd-Zvp and GAd-Eecto-induced polyclonal antibodies from sera. Binding to ZIKV Eecto, DIII, or DIII-LR mutant (A310E and T335K) was determined by ELISA from sera obtained post-boost at day 42 (n = 17 per group, ANOVA with a Tukey’s post-test: ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001).(G-J) Cell-mediated immune responses were analyzed at 90–110 days post-initial immunization. Splenocytes were assayed for IFN-?+ ZIKV E-specific CD8+ T cells using intracellular staining after peptide restimulation. Representative flow cytometry plots of four experiments are shown with percentages of IFN-?+ ZIKV-specific CD8+ T cells (G). Summary of frequencies of IFN-?+ ZIKV-specific CD8+ T cells are shown (H). ZIKV E-specific MBCs per million CD19+ cells obtained from the spleen were determined by ELISA and Poisson distribution analysis (see STAR Methods) (I). An ELISPOT determined the number of ZIKV-specific IgG producing LLPCs per million bone marrow cells (J). Bars indicate median values per vaccine group. Data for cell mediated responses are pooled from four experiments with n = 6–17 animal per group (ANOVA with a Dunnett’s post-test comparing vaccine and control groups: ns, not significant; **p < 0.01; ***p < 0.001). In this figure, dotted lines represent the limit of detection (LOD) of the assays.

Fig 5: Ex vivo flow cytometry analysis of T-B cell conjugates in Bcl6fl/flCD4-cre+/- and SAP-deficient mice during P. chabaudi infection. Bcl6fl/flCD4-cre+/- and Sh2d1a-/- mice were infected with P. chabaudi via intra peritoneal injection of 105 iRBC, and the occurrence of T-B cell conjugate in the spleen was analyzed ex vivo by flow cytometry at days 0, 15 and 35 post-infection. (a) Representative overlay histograms showing FSC-A for CD3+ CD4+ T cell singlets, B220+ CD19+ B cell singlets, and CD3+ CD4+ B220+ CD19+ T-B cell conjugates, obtained from the spleen of a wt C57BL/6 control mouse. (b) Representative plots of naïve (top row) and infected (35 days post-infection, bottom row) of Sh2d1a-/- and wt C57BL/6 control mice. Gates show frequency of CD3+ B220+ cells positive for CD19 and CD4. (c) Total numbers of CD3+ CD4+ B220+ CD19+ T-B cell conjugates in Sh2d1a-/- and wt C57BL/6 control mice. (d) Representative plots of naïve (top row) and infected (35 days post-infection, bottom row) of Sh2d1a-/- and wt C57BL/6 control mice. Gates show frequency of CD3+ CD4+ B220+ CD19+ T-B cell conjugates expressing low CD38 and high GL-7. (e) Total numbers of CD3+ CD4+ B220+ CD19+ CD38loGL-7+ T-B cell conjugates in Sh2d1a-/- and wt C57BL/6 control mice. (f) Representative plots of naïve (top row) and infected (35 days post-infection, bottom row) of Bcl6fl/flCD4-cre+/- and wt control mice. Gates show frequency of CD3+ B220+ cells positive for CD19 and CD4. (g) Total numbers of CD3+ CD4+ B220+ CD19+ T-B cell conjugates in Bcl6fl/flCD4-cre+/- and wt control mice. (h) Representative plots of naïve (top row) and infected (35 days post-infection, bottom row) of Bcl6fl/flCD4-cre+/- and wt control mice. Gates show frequency of CD3+ CD4+ B220+ CD19+ T-B cell conjugates expressing low CD38 and high GL-7. (i) Total numbers of CD3+ CD4+ B220+ CD19+ CD38loGL-7+ T-B cell conjugates in Sh2d1a-/- and wt C57BL/6 control mice. Bars represent median values. Data are representative or pooled from at least two independent experiments and were obtained in groups of 3–8 mice per time point. Statistical significance was obtained using Two-Way ANOVA with Tukey's multiple comparisons test. ND: Not done.