DOI: 10.5061/dryad.00000009x
title: Data from: Vaccine targeting to mucosal lymphoid tissues promotes humoral immunity in the gastrointestinal tract

description: Viruses, bacteria, and parasites frequently cause infections in the
 gastrointestinal (GI) tract, but traditional vaccination strategies
 typically elicit little or no mucosal antibody responses. Here, we report
 a strategy to effectively concentrate immunogens and adjuvants in
 gut-draining lymph nodes (LNs) to induce gut-associated mucosal immunity.
 We prepared nanoemulsions (NEs) based on biodegradable oils commonly used
 as vaccine adjuvants, which encapsulated a potent Toll-like receptor
 agonist and displayed antigen conjugated to their surface. Following
 intraperitoneal administration, these NEs accumulated in gut-draining
 mesenteric LNs, priming strong germinal center responses and promoting B
 cell class switching to IgA. Optimized NEs elicited 10- to 1000-fold
 higher antigen-specific IgG and IgA titers in the serum and feces,
 respectively, compared to free antigen mixed with NE, and strong
 neutralizing antibody titers against SARS-CoV-2. Thus, robust gut humoral
 immunity can be elicited by exploiting the unique lymphatic collection
 pathways of the gut with a lymph-targeting vaccine formulation.
description: # Data from: Vaccine targeting to mucosal lymphoid tissues promotes
 humoral immunity in the gastrointestinal tract
 [https://doi.org/10.5061/dryad.00000009x](https://doi.org/10.5061/dryad.00000009x) This dataset contains the raw data to plot the graphs shown in the paper. ## Description of the data and file structure Here, we report a strategy to effectively concentrate immunogens and adjuvants in gut-draining lymph nodes (LNs) to induce gut-associated mucosal immunity by using nanoemulsions (NEs) based on biodegradable oils commonly used as vaccine adjuvants, which encapsulated a potent Toll-like receptor agonist and displayed antigen conjugated to their surface. ## Files and variables ### File: Figure1-raw_data.csv **Description:** This dataset contains SEC profiles of eOD-Chol-NE and eOD-DSPE-NE together with unconjugated eOD-GT8. SEC experiments were conducted with fluorescent NE carrying BODIPY-cholesteryl ester and AF647-conjugated eOD-GT8. The dataset also contains DLS analysis (size distribution by volume) of eOD-Chol-NE and eOD-DSPE-NE together with unconjugated eOD-GT8 and NEs. PDI, polydispersity index. ##### Variables * **Normalized Fluorescence**: fluorescence signal for each fraction collected by size-exclusion chromatography * **% Frequency (volume)**: size distribution by volume of indicated nanoemulsions ### File: Figure2-raw_data.csv **Description:** This dataset contains quantification [(B), (D), and (F)] of fluorescence from AF647-labeled eOD in excised lymph nodes 6 hours following intravenous (iv) [(B)], subcutaneous (sc) [(D)], or intraperitoneal (ip) [(F)] injection of eOD-Chol-NE, eOD-DSPE-NE, or eOD/NE (10 μg of eOD, 5 μg of 3M-052, and 0.5 μg of squalene NE per vaccine dose, n= 4 mice per group. The dataset also contains mean NE fluorescence signals of NE (H) and eOD antigen (J) over time. ##### Variables * **Total Radiant Efficiency [p/s] / [μW/cm^2]**: quantitative readout of fluorescence emission from excised lymph nodes ### File: Figure3-raw_data.csv **Description:** This dataset contains the percentage of eOD-GT8 or NE-positive splenocytes, and quantification of the mean frequency of eOD-GT8+VRC01+ double-positive cells, after cells were incubated with fluorescently labeled eOD-Chol-NE, eOD-DSPE-NE, or eOD/NE at the indicated concentrations. ##### Variables * **% eOD+**: percent of splenocytes with positive signal for fluorescent eOD-GT8 measured by flow cytometry * **% NE+**: percent of splenocytes with positive fluorescent signal for nanoemulsion measured by flow cytometry * **% eOD+/VRC01+**: percent of splenocytes with positive signal for fluorescent eOD-GT8 and able to bind to VRC01 antibody as measured by flow cytometry ### File: Figure4-raw_data.csv **Description:** This dataset contains GC responses in mesenteric LNs  (mesLNs) analyzed by flow cytometry at day 12 after mice (n=5 mice per group) were immunized with eOD-Chol-NE, eOD-DSPE-NE, or eoD/NE (10 μg of eOD-GT8, 5 μg of 3M-052, and 0.1 μg of squalene NE). The dataset also contains anti-eOD antibody titers in serum and feces of immunized animals measured over time by ELISA. ##### Variables * **%PD-1+ CXCR5+ TfH cells**: T-follicular helper cells were quantified by flow cytometry and identified as CD19-, CD4+, CD44+, PD-1+, and CXCR5+ * **%GC B cells**: Germinal center B cells were quantified by flow cytometry and identified as CD19+, CD38-, GL7+ * **%eOD+ of GC B cells**: percentage of GC B cells, as designated above, that were able to bind to eOD-GT8 tetramers, detected by flow cytometry * **%IgA+ of eOD+ GC B cells**: percentage of eOD+ GC B cells, as designated above, that expressed IgA on their surface, detected by flow cytometry * **log titers**: log transformations of endpoint anti-eOD antibody titers measured by ELISA ### File: Figure5-raw_data.csv **Description:** This dataset contains anti-SARS-CoV-2 RBDJ or anti-SARS-CoV-2 S1 antibody titers (IgG and IgA) and SARS-CoV-2 pseudovirus neutralizing ID~50~ titers (PSVNT50) in serum and feces of immunized animals measured by ELISA. ##### Variables * **log titers**: log transformations of end point antibody titers anti-RBDJ or anti-S1 from indicated SARS-CoV-2 variants, measured by ELISA * **PSVNT50**: pseudovirus neutralizing ID~50~ titers, which was determined as the dilution of the sample at which there was a 50% decrease in SARS-CoV-2 pseudovirus (GFP)-positive cells compared to the average of the virus control wells. ### File: FigureS1-raw_data.csv **Description:** This dataset contains UV-Vis absorbance curve of DBCO-PEG12-Maleimide modified eOD-GT8. ##### Variables * **Absorbance**: absorbance of DBCO-PEG12-Maleimide modified eOD-GT8 at indicated wavelengths ### File: FigureS2-raw_data.csv **Description:** Characterization of antigen-conjugated nanoemulsions: (C and D) Absorbance curves of the Chol-NE (C) and DSPE-NE (D) with and without 3M-052. The peak at ~320 nm is specific to 3M-052. (E and F) Size exclusion chromatography (SEC) profiles of Chol-NE (E) and DSPE-NE (F) showing 3M-052 incorporation in the NEs. For SEC analysis, BODIPY-cholesteryl ester was encapsulated in the NEs, and the absorbance wavelength of the dye (at 510 nm) was used to trace the NEs together with the absorbance wavelength of 3M-052. (H) DLS analysis (size distribution by intensity) of eOD-Chol-NE and eOD-DSPE-NE. ##### Variables * **Absorbance**: absorbance of nanoemulsions at indicated wavelengths * **Normalized absorbance**: absorbance measurement of each nanoemulsion fraction collected by size-exclusion chromatography * **% Frequency (volume)**: size distribution by volume of indicated nanoemulsions ### File: FigureS3-raw_data.csv **Description:** Size exclusion chromatography (SEC) profile of eOD-GT8 with cholesterol tail and eOD-GT8 accumulation in the mesenteric lymph nodes as measured by in vivo imaging (IVIS) ##### Variables * **Normalized Fluorescence**: fluorescence signal for each fraction collected by size-exclusion chromatography * **Total Radiant Efficiency [p/s] / [μW/cm^2]**: quantitative readout of fluorescence emission from excised lymph nodes by IVIS ### File: FigureS4-raw_data.csv **Description:** In-vitro antigen transfer from NEs to the splenocytes in the FBS-free RPMI medium, measured by flow cytometry. Splenocytes were incubated with eOD-Chol-NE, eOD-DSPE-NE, or eOD/NE for 1 hour, in FBS-free medium at 37°C at a range of concentrations, then washed and stained with VRC01-PE antibody. ##### Variables * **% eOD+/VRC01+ splenocytes**: percent of splenocytes with positive signal for fluorescent eOD-GT8 and able to bind to VRC01 antibody as measured by flow cytometry ### File: FigureS5-raw_data.csv **Description:** In-vivo eOD-GT8/NE uptake by T cells. Mice were immunized with fluorescent eOD-Chol-NE, eOD-DSPE-NE, or eOD/NE. Mesenteric lymph nodes were harvested 24h later for flow cytometry analysis of antigen uptake. ##### Variables * **% eOD+**: percent of CD19-, CD3+ T cells with positive eOD-GT8-AF647 fluorescent signal, as measured by flow cytometry * **% NE+**: percent of CD19-, CD3+ T cells with positive NE (BODIPY) fluorescent signal, as measured by flow cytometry ### File: FigureS7-raw_data.csv **Description:** This dataset contains anti-eOD antibody ELISA absorbance vs dilution curves at week 6 post-immunization for serum IgG (A), serum IgA (B), fecal IgG (C), and fecal IgA (D). Mice (n = 5 per group) were immunized with eOD-Chol-NE, eOD-DSPE-N,E or eOD/NE and boosted 4 weeks later. ##### Variables * **ELISA Absorbance**: absorbance of serum or fecal samples processed by ELISA at indicated dilutions at 450 nm with 540-nm reference ### File: FigureS8-raw_data.csv **Description:** This dataset contains anti-eOD antibody titers in serum and feces of animals immunized either via intraperitoneal (i.p.) or subcutaneous (s.c.), measured over time by ELISA. Mice (n=5 per group) were immunized with eOD-Chol-NE and boosted 4 weeks later. ##### Variables * **log titers**: log transformations of endpoint anti-eOD antibody titers measured by ELISA at indicated time points ### File: FigureS9-raw_data.csv **Description:** This dataset contains GC responses in mesLNs analyzed by flow cytometry at day 12 after mice (n=5 mice per group) were immunized with eOD-Chol-NE with or without the adjuvant 3M-052. The dataset also contains anti-eOD antibody titers (IgG and IgA) in serum and feces of immunized animals measured by ELISA. ##### Variables * **%GC B cells**: Germinal center B cells were quantified by flow cytometry and identified as CD19+, CD38-, GL7+ * **%eOD+ of GC B cells**: percentage of GC B cells, as designated above, that were able to bind to eOD-GT8 tetramers, detected by flow cytometry * **%IgA+ of eOD+ GC B cells**: percentage of eOD+ GC B cells, as designated above, that expressed IgA on their surface, detected by flow cytometry * **log titers**: log transformations of endpoint anti-eOD antibody titers measured by ELISA ### File: FigureS10-raw_data.csv **Description:** This dataset contains anti-SARS-CoV-2 RBDJ antibody titers in bronchoalveolar lavage fluid (BALF), measured over time by ELISA. Mice (n = 5 per group) were immunized with RBDJ-Chol-NE, RBDJ-DSPE-NE, or RBDJ/NE and boosted 4 weeks later. BALF was collected from instillations of PBS (2 x 1 ml) supplemented with Complete protease inhibitor cocktail (Roche) in the lungs using a catheter through the trachea. ##### Variables * **log titers**: log transformations of endpoint anti-RBDJ antibody titers (IgG and IgA)measured by ELISA