Fig 2: Eosinophil behavior and distribution in the lung following ovalbumin sensitization and challenge. EoCRE+/-/tdTomato+/- mice were injected with either phosphate-buffered saline (PBS) or ovalbumin (Ova) and then challenged with aerosolized Ova as described in the “Methods.” Forty-eight hours after the last Ova challenge, mice were prepared for intravital microscopy of the lung. a The migration tracks of eosinophils from either PBS/Ova (PBS) or Ova/Ova (Ova) animals are shown. The number of crawling eosinophils (b) and the distance traveled (c) were measured. Data in b are mean ± SEM and individual values from n = 5 independent experiments (PBS) or n = 6 independent experiments (Ova) (*p = 0.0173) and the data in c show the individual measurements of track length. d, e Prior to intravital imaging of eoCRE+/-/tdTomato+/- mice, animals were given an intravenous injection of anti-CD45 conjugated to Alexa-647. Images from individual channels are shown in grayscale. Merged images show eosinophils in green, intravascular leukocytes labeled with anti-CD45 antibody in magenta, and the vasculature in blue. Data represent three independent experiments. f Dispersed eosinophils or eosinophils in clusters are shown in f and the extent of clustering is quantified in g. Data in g represent the mean ± SEM of n = 5 independent experiments (PBS) or n = 6 independent experiments (Ova). *p = 0.0152. h Lungs from Ova/Ova eoCRE+/-/tdTomato+/- mice were fixed and imaged as described in “Methods.” Scale bars are 50 µm

Fig 3: Selective IGHV1-2*02 B cell lineage expansion using the engineered vaccine immunogen VLP F2. (A) CD45.1+/+ mice received IGHV1-2*02 IgM B cells (1x106 B cells per recipient) and were then either unimmunized, immunized with control VLP or immunized with VLP F2. CD45.1 vs CD45.2 reactivity is shown for CD3-/CD19+ B cells in each group. (B) CD45.1 vs CD45.2 reactivity was then evaluated within the plasmablast gate (CD19int/CD138+). (C) IGHV1-2*02 B cells expanded to plasmablasts in animals immunized with control VLP or VLP F2 (mean ± SD, n = 5 per group, standardized as the proportion of the total IgM IGHV1-2*02 B cells transferred to each recipient animal ***P < 0.001, Student’s T test). (D) Extrafollicular and follicular pathways for B cell activation and expansion following immunization, starting from the naïve IgM BCR stage through to differentiation to plasmablasts and plasma cells.

Fig 4: The bispecific iv4/iv9 molecule activates iGL-VRC01 B cells in vivo500,000 purified, CellTrace Violet (CTV)-labeled B cells from homozygous iGL-VRC01 CD45.2+ mice were transferred into wild-type recipient CD45.1+ mice. The next day, mice were immunized with adjuvant plus 10 µg of iv4-SC, iv9-ST, iv4/iv9, or with adjuvant alone mixed in PBS. Splenocytes and serum were harvested 5 or 14 days after immunization, and the B cell response was analyzed by flow cytometry and ELISA.(A) The precursor frequency of live iGL-VRC01 B cells at the time of immunization (24 h after injection) was assessed in two separate experiments represented by white and black dots.(B and C) The proliferation of antigen-specific (iv4/iv9+) B cells upon immunization was assessed by analyzing the intensity of CTV staining to determine the percentage of cells that had divided 5 days after immunization.(D and E) Frequencies of total CD45.2+ B cells after 5 days (D) and 14 days (E).(F) Frequency of antigen-specific (iv4/iv9+) CD45.2+ B cells in germinal centers (GC; CD45.2+iv4/iv9+, Bcl6+GL7+) at day 14.(G) Gating strategy to identify antigen-specific GC (Bcl6+GL7+) B cells that are endogenous (CD45.1+) or transferred (CD45.2+).(H) Frequency of antigen-specific GC B cells that were recruited from CD45.1+ or CD45.2+ B cells at day 14.(I and J) Total IgG binding to eOD-GT8 (solid circles) and eOD-GT8 knockout (KO) (open circles, dashed lines) in serum of animals 14 days after immunization as measured by ELISA. (I) PBS immunized. (J) iv4/iv9 immunized.Results are shown as mean ± SD, with each circle representing a single animal. The p values were determined by one-way ANOVA and a Tukey’s multiple comparison test (day 5) or Student’s t test (day 14). *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 5: Method overview. (A) A highly immunogenic RNA bacteriophage VLP platform has been engineered to display diverse guest peptide sequences. Each VLP encapsidates its own mRNA, meaning that the VLPs can be deployed for affinity selection. The library is then interrogated for specificity to antibodies with chimeric antigen binding sites, serving to identify VLP bearing affinity to a specific antibody feature (in this study the human IGHV1-2*02 domain). (B) The identified VLP is then verified by antibody binding and its capacity to trigger the BCR configuration of interest when displayed by a reporter B cell line. (C) To evaluate the corresponding B cell lineage expansion in vivo, transgenic human-like CD45.2+/+ BCRs constrained to the motif are adoptively transferred to recipient CD45.1+/+ C57Bl/6 mice. In this case the adoptively transferred B cells are constrained in IGHV1-2*02 usage, but unconstrained all other BCR features and bear human-like diversity in CDRH3 (31, 53).