DOI: 10.5061/dryad.n2z34tn7k
title: Data from: DNA origami vaccines program antigen-focused germinal centers

description: Priming rare subdominant precursor B cells in germinal centers (GCs) is a
 central goal of vaccination to generate broadly neutralizing antibodies
 (bnAbs) against HIV. Multivalent immunogen display on protein nanoparticle
 scaffolds can promote such responses, but it also generates scaffold-
 specific B cells that could theoretically limit bnAb precursor expansion
 in GCs. We rationally designed DNA origami–based virus–like particles
 (DNA- VLPs) displaying a germline- targeting HIV envelope protein
 immunogen, which elicited no scaffold-specific antibody responses.
 Compared with a state- of- the- art clinical protein nanoparticle, these
 DNA- VLPs increased the expansion of epitope-specific GC B cells relative
 to off-target B cells and enhanced expansion of bnAb- lineage B cells in a
 humanized mouse model of CD4 binding site priming. Thus, minimizing
 off-target responses enhances bnAb priming and indicate DNA- VLPs are a
 promising vaccine platform.
description: <strong>For complete methods, please see accompanying
 article (Romanov et. al. 2026.)</strong>
 <strong>DNA-VLP synthesis and
 characterization</strong> DNA-VLPs were assembled
 as previously described(<em>53</em>). All designs were
 generated using DAEDALUS(<em>53</em>) (d40 VLP) or
 ATHENA(<em>49</em>) (d30 VLP) with staples manually adjusted
 in Tiamat software(<em>87</em>) to have outward nick positions
 (2 per edge). To fold origami, 30 nM of scaffold was mixed with 5–10x
 excess of each oligonucleotide staple in TAE buffer with 12 mM
 MgCl<sub>2</sub> and thermally annealed as follows: 95°C for 5
 min, 80–75°C at 1°C per 5 min, 75–30°C at 1°C per 15 min, and 30–25°C at
 1°C per 10 min. DNA-VLPs**** were purified into PBS using Amicon Ultra 100
 kDa centrifugal filters (Millipore Sigma, cat #UFC810024) spun at 2000g
 and stored at 4 °C. Purity and dispersity of DNA-VLPs were validated by
 AGE (1.6% agarose, TAE buffer with 12 mM MgCl<sub>2</sub>,
 SYBR Safe, 65V for 150 min) and dynamic light scattering. For antigen
 conjugation, DNA-VLPs were concentrated to &gt;1 μM and reacted with
 at least 3 molar equivalents of eOD-Azide for 24 hours at 37°C. Excess
 antigen was removed by drop dialysis into 1X PBS for 16 hours (Millipore
 Sigma, mixed cellulose ester, 0.025 µm) and the purified nanoparticles
 were diluted to 100 nM and stored at 4°C. Antigen functionalization was
 validated using AGE (1.6% agarose, TAE buffer with 12 mM
 MgCl<sub>2</sub>, SYBR Safe, 65V for 150 min) and quantified
 using bicinchoninic acid colorimetric assay.
 <strong>Negative stain transmission electron
 microscopy</strong> Uranyl formate staining was
 performed as previously described(<em>28</em>). Briefly,
 DNA-VLPs were diluted to 3–5 nM in PBS and 5 μL was applied onto
 glow-discharged electron microscopy grids. After 30 seconds, the grids
 were blotted with filter paper and washed with 5 μL of fresh 2% uranyl
 formate solution containing 5 mM NaOH for 30 seconds. The uranyl formate
 solution was removed by blotting on filter paper, and the grids were
 stored with a desiccant before imaging. TEM was performed on an FEI Tecnai
 G2 Spirit Twin. Shown are images at 30K, 42K, or 67K
 magnifications. <strong>CryoEM
 imaging</strong> Three microliters of the folded
 and purified DNA-VLP solution (approximately 300 nM) was applied onto the
 glow-discharged 200-mesh Quantifoil 2/1 grid, blotted for four seconds and
 rapidly frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher
 Scientific). Grids were screened and imaged on a Talos Arctica
 cryo-electron microscope (Thermo Fisher Scientific) operated at 200 kV at
 a magnification of 79,000× (corresponding to a calibrated sampling of 1.76
 Å per pixel). Micrographs were recorded by EPU software (Thermo Fisher
 Scientific) with a Gatan K2 Summit direct electron detector in counting
 mode, where each image is composed of 24 individual frames with an
 exposure time of 6 s and a total dose ~63 electrons per Å2. We used a
 defocus range of –1.0 – –2.5 μm to collect images, which were subsequently
 motion-corrected using MotionCor2. Particle picking was performed manually
 using Relion(<em>88</em>) with a particle box size of
 320. <strong>Flow cytometry analysis of GC B and
 Tfh cells</strong> Mice were euthanized at the
 indicated time point post-injection by CO<sub>2</sub>
 asphyxiation, and inguinal lymph nodes were collected and mechanically
 digested into a single-cell suspension using Biomasher tubes and a
 motorized tissue grinder. Lymphocytes were strained twice through 60 μm
 Multi-Screen Mesh filter plates (Millipore Sigma, cat# MANMN6010) and
 washed once in 1X PBS. The cells were then stained with 1:750 dilution of
 Zombie UV Live Dead Stain (BioLegend, cat# 423107) diluted in PBS for 10
 minutes at room temperature. Excess dye was removed by washing once with
 FACS buffer (1X PBS, 2% FBS, 0.01% sodium azide). The cells were then
 resuspended in 25 μL FACS buffer containing Fc block (BioLegend, cat#
 156603) and incubated on ice for 15 minutes. Antibody mixes (2X stock)
 were prepared in FACS buffer and contained the following antibodies:
 anti-CD4 BUV737 (BD Biosciences, RM4.5), anti-B220 APC-Cy7 or PE-Cy7
 (BioLegend, RA3-6B2), anti-CD38-AF488 or BV421 (BioLegend, 90), anti-GL7
 PerCpCy5.5 (BioLegend, GL7), anti-CXCR5 BV605 (BioLegend, L138D7),
 anti-PD1-AF647 or BV421 (BioLegend, 29F.1A12), and antigen tetramer
 probes. For the experiment shown in Fig. 5, anti-IgM-PE-Cy7 (BioLegend,
 MA-69) and anti-IgD-BV785 (BioLegend, 11-26c.2a) were also included in the
 antibody cocktail. Tetramers were added to antibody mix so that the final
 amount per sample was 50 ng. For nanoparticle probes (Fig 5 & 6),
 nanoparticle probes were added at 5 ng/ sample. The cells in Fc block were
 mixed with antibody and probe mixture and incubated for 30 minutes on ice,
 then washed twice with FACS buffer. The cells were fixed with 2% PFA,
 washed once and stored in FACS buffer until flow cytometry analysis. The
 cells were transferred to U-bottom 96-well plates and mixed with
 CountBright Absolute Counting beads (Thermo Fisher Scientific, cat#
 C36950) before analysis. Flow cytometry was carried out on a BD LSR
 Fortessa or Symphony A3 cytometer in plate-mode. Full-minus-one (FMO)
 staining controls were included in every experiment for drawing
 gates. <strong>Lymph node
 microscopy</strong> For lymph node histology,
 mice were immunized with 5 μg of fluorescently labeled eOD-GT8 monomer,
 DNA-VLPs, or protein nanoparticles and sacrificed at the indicated time
 points. Mice were euthanized by CO<sub>2</sub> asphyxiation,
 and inguinal lymph nodes were flash frozen in OCT embedding medium and
 sectioned on a Leica Cryostat (10 μm thick sections) and stored at –80°C.
 For immunostaining, the sections were quickly thawed, fixed with 10%
 neutral buffered formalin, and blocked/permeabilized with PBS containing
 2% BSA and 0.01% Triton-X. The sections were then stained with 1:100
 dilutions of anti-CD35-BV421 (BD Biosciences, clone 8c12),
 anti-CD169-AF488 (BioLegend clone 3D6.112), or anti-F4/80 (BioLegend clone
 BM8) in block/perm buffer for 1 hour in a humidity chamber. The slides
 were washed three times in PBS and mounted with ProLong Diamond Antifade
 mountant (Thermo Fisher Scientific) and secured with sealant. Slides were
 stored in the dark and imaged on a Leica Sp8 Laser Scanning Confocal
 Microscope with 25x water-based objective. Colocalization between channels
 was quantified using a MATLAB script which applied a Gaussian filter to
 each channel, created a binary mask to identify FDC or SSMs, and
 calculated the fraction of area occupied by antigen signal. In ImageJ, LUT
 tables were selected to optimize contrast and enhance readability for
 color-blindness. For cleared lymph node imaging, mice
 were immunized with 5 or 10 μg of fluorescently labeled nanoparticles
 (specified in captions). For in situ follicle labeling, 4 μg of
 anti-CD35-BV421 (BD, clone 8c12) was injected subcutaneously 12–16 hours
 prior to tissue harvesting. Inguinal lymph nodes were isolated and fixed
 in 4% PFA for 24 hours and cleared using DISCO as previously
 described(<em>61</em>). Briefly, LNs were washed twice in PBS
 and excess fat and connective tissue were removed. Nodes were then
 gradually moved into solutions containing successively higher
 concentrations of methanol until they were incubated for half an hour in
 pure methanol. Nodes were then bleached in hydrogen peroxide for one
 minute before being returned to methanol for half an hour. They were then
 gradually transferred into solutions containing increasing concentrations
 of tertiary-butanol before eventually being incubated in pure
 tertiary-butanol 0.4% α-tocopherol for one hour. Nodes were then removed
 from solution and allowed to dry completely before being placed in
 dichloromethane. After the lymph nodes dropped to the bottom of tubes
 following swirling, they were stored in dibenzyl ether with 0.4%
 α-tocopherol. Follicles were imaged using an Olympus FV1200 Laser Scanning
 Confocal Microscope at 10x magnification over a 300 μm distance. Lasers
 were set to minimize pixel saturation in the brightest samples. Images
 were analyzed using ImageJ software. LUT tables were selected to optimize
 contrast and enhance interpretability for color-blindness.
 <strong>Single cell BCR sequencing</strong>
 CD4bs-specific GC B cells were FACS-sorted from inguinal lymph
 nodes harvested from VH1-2 mice immunized with protein or DNA-scaffolded
 immunogens, gated on
 B220<sup>+</sup>/CD38<sup>lo</sup>GL7<sup>hi</sup>/IgM<sup>-<sup>IgG</sup>+</sup>/eOD<sup>++<sup>KO</sup>-</sup> populations. We generated BCR libraries from whole transcriptome amplification (WTA) products produced using the Smart-Seq2 protocol on the FACS isolated B cells(<em>89</em>). The WTA products underwent two 0.8x (v/v) SPRI bead-based cleanups and were verified using High Sensitivity D5000 ScreenTape (Agilent Technologies Inc) and quantified and normalized using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, cat # Q32854). To enrich the BCR sequences (FR1 to CDR3), corresponding heavy and light chains were amplified (HotStarTaq Plus Master Kit, Qiagen, cat #203645) with a pool of partially degenerate primers specific against all possible IGHV (human) or IGLV (mouse) and IGKV (mouse) segments in the FR1 region (final concentration: 10 µM each) and reverse primers against the heavy or light constant regions (final concentration: 10 µM each)(<em>90</em>). These primers were also built with attachments to the Illumina P7 (V region) and P5 (constant region) sequences(<em>90</em>). The amplicons were quantified and normalized following BCR amplification and SPRI cleanup, after which cellular barcodes and Illumina sequencing adapters (Nextera XT Index Adapters, Illumina Inc.) were added to each amplified heavy and light chain using step-out PCR (Kapa HiFi HotStart ReadyMix; Fisher Scientific cat # 50-196-5217). After another SPRI cleanup the HC and LC samples were pooled and the single-cell BCR libraries were sequenced via paired-end 250x250 reads and 8x8 index reads on an Illumina MiSeq System (MiSeq Reagent Kit v2 (500-cycle), cat# MS-102-2003). The BCR heavy and light chains reads were then paired using the barcodes and the overlapping sequencing reads were reconstructed with PandaSeq(<em>91</em>), and aligned against the human or mouse IMGT database(<em>92</em>).  Sequencing error correction was performed with MigMAP, a wrapper for IgBlast (https://github.com/mikessh/migmap). Consensus V<sub>H</sub> and V<sub>L</sub>/V<sub>K</sub> chain for each single cell was achieved by collating all reads with the same CDR3 sequence and then calling the top heavy and light chain sequences by frequency. The nucleotide sequences (full-length heavy chain sequences) from the public BCRs bearing the short five amino acid CDRL3 were aligned with unmutated germline (IGHV1-2*02) sequence using the ClustalW alignment tool in the MEGAx(<em>93</em>). The phylogenetic trees were constructed using the neighbor-joining method with Poisson Model in the MEGAx(<em>94</em>). The reliability of the tree nodes was tested using the Felsenstein bootstrap method with 500 replicates. <strong>Statistics</strong> Statistics were computed in GraphPad Prism v.9 as denoted in the figure captions. For flow cytometry analyses, comparisons between two groups were made using a two-sided Student’s t-test or Mann Whitney U test if the sample size was too small to pass a normality test. For experiments with comparison with 3 or more groups, we used one-way analysis of variance (ANOVA) with host-hoc Tukey test for multiple comparisons. For data shown on log axes, geometric means and geometric S.D. values are shown. No outliers were excluded. One mouse from Fig.4 was excluded due to accidental pregnancy. Exact <em>P</em> values are denoted in the figures. For all figures, NS is not significant (<em>P</em> &gt; 0.05).
description: # Data from: DNA origami vaccines program antigen-focused germinal centers
 Dataset DOI: [10.5061/dryad.n2z34tn7k](10.5061/dryad.n2z34tn7k) Full
 article author llist: Anna Romanov, Grant A. Knappe, Larance Ronsard,
 Christopher A. Cottrell, Yiming J. Zhang, Heikyung Suh, Lauren Duhamel,
 Marjan Omer, Asheley P. Chapman, Katie Spivakovsky, Patrick Skog, Claudia
 T. Finn, Jeong Hyun Lee, Oleksandr Kalyuzhniy, Alessia Liguori, Molly F.
 Parsons, Vanessa R. Lewis, Josue Canales, Boris Reizis, Ryan D. Tingle,
 Torben Schiffner, William R. Schief, Daniel Lingwood, Mark Bathe, Darrell
 J. Irvine Contacts: Anna Romanov (first author):
 [romanov@mit.edu](mailto:romanov@mit.edu) Darrell J. Irvine (corresponding
 author): [djirvine@scripps.edu](mailto:djirvine@scripps.edu) Mark Bathe
 (corresponding author): [mark.bathe@mit.edu](mailto:mark.bathe@mit.edu)
 Date of data collection: 2021–2025 Location of data collection: MIT
 Biological Engineering, MIT Nano, MIT BioMicro Center, Koch Institute for
 Integrative Cancer Research, Ragon Institute of MGH, MIT and Harvard,
 Scripps Research Institute.  ## Description of the data Raw data files,
 including FCS files (flow cytometry), electron microscopy micrographs,
 confocal microscopy images, and agarose gel electrophoresis images are
 provided in the .zip files corresponding to each figure. FCS are
 uncompensated (compensation calculated during analysis in Flowjo) with
 compensation controls  and panel descriptions provided with each dataset.
 ### Files and variables #### **File: Fig_1.zip** **Title/Description:
 Nanoparticulate eOD-GT8 assembly on icosahedral DNA-VLP scaffolds.** This
 folder contains data supporting the characterization of d40-30mer
 DNA-VLPs, including raw electrophoresis data and flow cytometry data for
 characterization of germinal center responses in mice after immunization
 with DNA-VLPs or eOD protein control. This folder contains the following
 subfolders: /*AGE*. This subfolder contains raw gel electrophoresis data
 that shows conjugation of eOD onto d40-VLPs (assessed by band shift)
 (.tif); /*FCS-eOD vs d40-30mer.*  This subfolder contains flow cytometry
 data (.fcs) including subfolders containing sample files (FCS-d40-30mer
 and FCS-monomer) with 01-05 denoting individual mice, full-minus-one
 controls (FCS-FMOs), single color controls used for compensation
 (FSC-compensation controls), the exact compensation matrix used for
 analysis (compensation matrix.csv), and description of the flow panel
 including makers and fluorophores (panel info.txt) used to quantify
 germinal center responses in response to immunization with d40-30mer or
 eOD monomer.   /*FCS-DNase WT vs KO.* This folder contains flow cytometry
 data (.fcs) including sample files (labeled with the mouse strain and
 biological replicate), single color controls used for compensation
 (labeled compensation control followed by fluorophore), the exact
 compensation matrix used for analysis (compensation matrix.csv), and
 description of the flow panel including makers and fluorophores (panel
 info.txt) used to quantify germinal center responses in response to
 immunization with DNA-VLP in DNase I KO or DNase I WT mice. #### **File:
 Fig_2.zip** **Title/Description:** **d40-30mer DNA-VLPs are poorly
 retained on follicular dendritic cells in draining lymph nodes.** This
 folder contains confocal microscopy data (.tif) used characterize DNA-VLP
 compared to protein trafficking in lymph nodes. The file contains the
 following subfolders:  /*IHC- Whole LNs*. This subfolder contains raw
 8-bit .tif files and RGB files with confocal microscopy data collected on
 Leica Sp8, 25x objective. Images are saved in subfolders (eOD-GT8 monomer,
 d40-30mer, p60mer),  based on which immunogen is shown. Microscopy image
 analysis was performed to assess the spatial distribution of antigen
 signal relative to follicular dendritic cell (FDC) and subcapsular sinus
 macrophage (SSM) networks to account for the fact that images were
 collected across different acquisition sessions. Multichannel fluorescence
 images were separated into individual channels corresponding to antigen,
 FDC, and SSM signals. Each channel was minimally smoothed to reduce
 pixel-level noise, and binary masks were generated using fixed intensity
 thresholds chosen based on background signal levels and applied
 identically across all images. Small isolated objects were removed to
 reduce segmentation noise. Antigen occupancy within FDC or SSM regions was
 quantified as the fraction of FDC- or SSM-positive pixels that overlapped
 with antigen-positive pixels. This analysis provides a comparative measure
 of spatial overlap across samples and does not rely on intensity
 correlation metrics or subcellular colocalization analyses. The resulting
 occupancy values are reported in the occupancy_results.csv file. 
 /*Cleared LNs.* This folder contains two .tiff image files corresponding
 to the images used in the manuscript for qualitative assessment of antigen
 signal (red) in follicles (blue). Each image is labeled according to the
 immunogen it represents. #### File: Fig_3.zip
 **Title/Description:** **High density antigen display enhances lectin
 pathway activation, FDC targeting, and antigen-specific GC B cell
 responses.** This folder contains confocal microscopy data (.tif), flow
 cytometry data (.fcs), compensation (.csv), and a description of flow
 panel (.txt). These data were generated to characterize immune responses
 produced by several different DNA-VLPs. */Follicle imaging.* This folder
 contains raw 8-bit tiff files (maximum projection stack) acquired on
 Olympus FV1200 using a 10x objective. The data is organized into
 subfolders based on which immunogen group was imaged (eOD-GT8 monomer,
 d30-30mer, d30-60mer, d40-30mer, d40-60mer, p60mer). Up to 20 different
 regions were imaged from 10 cleared lymph nodes from n=5 mice. Channel one
 represents CD35 staining and channel two is fluorescent antigen
 signal. Microscopy image analysis was performed to assess the spatial
 distribution of antigen signal relative to follicular dendritic cell
 (FDC). Multichannel fluorescence image stacks (maximum projection) were
 separated into individual channels corresponding to antigen and FDC
 signals. Each channel was minimally smoothed to reduce pixel-level noise,
 and binary masks were generated using fixed intensity thresholds chosen
 based on background signal levels and applied identically across all
 images. Antigen occupancy within FDC regions was quantified as the
 fraction of FDC-positive pixels that overlapped with antigen-positive
 pixels. This analysis provides a comparative measure of spatial overlap
 across samples and does not rely on intensity correlation metrics or
 subcellular colocalization analyses.  /*FCS files- GC B cell
 analysis*. This subfolder contains flow cytometry data (.fcs) including
 subfolders containing sample files  (e.g. FCS-d40-30mer) with 01-05
 denoting individual mice, full-minus-one controls (FCS-FMOs), single color
 controls used for compensation (FSC-compensation controls), the exact
 compensation matrix used for analysis (compensation matrix.csv), and
 description of the flow panel including makers and fluorophores (panel
 info.txt) used to quantify germinal center B cell responses in response to
 immunization with different DNA-VLPs.  */FCS files*- *Tfh analysis*. This
 subfolder contains flow cytometry data (.fcs) including subfolders
 containing sample files  (e.g. FSCd40-30mer) with 01-05 denoting
 individual mice, full-minus-one controls (FCS-FMOs), single color controls
 used for compensation (FSC-compensation controls), the exact compensation
 matrix used for analysis (compensation matrix.csv), and description of the
 flow panel including makers and fluorophores (panel info.txt) used to
 quantify Tfh cell responses in response to immunization with different
 DNA-VLPs. Note- this data is based on the same cell samples from the above
 GC B cell analysis, but the samples were split in half and stained
 separately with two antibody panels.  #### **File: Fig_4.zip**
 **Title/Description:** **Incorporating synthetic helper T cell epitopes
 boosts GC size after DNA-VLP vaccination.** This folder contains raw flow
 cytometry data (.fcs) including subfolders containing sample files  (e.g.
 /FSC-d30-60mer, /FSC-d30-60mer-PADRE) with 01-08 denoting individual mice,
 full-minus-one controls (/FCS-FMOs), single color controls used for
 compensation (/FSC-compensation controls), the exact compensation matrix
 used for analysis (compensation matrix.csv), and description of the flow
 panel including makers and fluorophores (panel info.txt) used to quantify
 GC B cell and Tfh cell responses in response to immunization with
 different DNA-VLPs. Replicates 1-8 represent a unique mouse. #### **File:
 Fig_5.zip** **Title/Description:** **DNA-VLPs produce focused germinal
 centers and prime VRC01-class precursors in humanized mice.** This folder
 contains flow cytometry (.fcs), compensation (.csv), description of flow
 panel (.txt) used to compare immune responses with different formulations
 in VH1-2 mice. The data is organized into subfolders based on the antigen
 probe used (/FCS- eOD 60mer probes, /FCS- DNA-VLP probes, /FCS-LumSyn
 probes). The data is further divided based on which immunogen was used to
 immunize the mouse (p60mer or d30-60mer-PADRE). Full-minus-one controls
 were used for establishing gates and files are found in */FCS-FMO*. Each
 experimental file is labeled 1-8 which represents a unique mouse. The
 exact compensation matrix used for analysis (compensation matrix.csv), and
 description of the flow panel including makers and fluorophores (panel
 info.txt) are provided. #### **File: Fig_6.zip**
 **Title/Description:** **BCR sequencing reveals DNA-60mers effectively
 prime VRC01-class precursors in early GCs.** This folder contains raw BCR
 sequencing reads (.fastq) used to generate paired heavy and light chain
 antibody sequences collected on Illumina NextSeq 500. The folders contain
 fastq files for DNA-VLP and p60mer groups for each mouse that are labelled
 for heavy and light chains separately. These fastq files were loaded and
 assembled by PandaSeq for BCR sequence reads using the barcodes and the
 overlapping sequencing reads were reconstructed which were aligned against
 the human or mouse IMGT database. MigMAP was used for processing heavy and
 light chain sequence alignment based on IgBlast to collect hypermutated
 antibody variants.  The folder contains the following subfolders:\ */1a.
 DNA_HC_mouse1*= DNA origami group Heavy chains from mouse 1. */1b.
 DNA_HC_mouse2* = DNA origami group Heavy chains from mouse 2. */2a.
 DNA_LC_mouse1* = DNA origami group Light chains from mouse 1. */2b.
 DNA_LC_mouse2* = DNA origami group Light chains from mouse 2. */3a.
 Protein_HC_mouse1* = Protein group Heavy chains from mouse 1. */3b.
 Protein_HC_mouse2* = Protein group Heavy chains from mouse 2. */4a.
 Protein_LC_mouse1* = Protein group Light chains from mouse 1. */4b.
 Protein_LC_mouse2* = Protein group Light chains from mouse2. 
 
 #### **File: Fig_S2.zip** **Title/Description: Characterization of d40
 and d30 DNA-VLP series.** This folder contains data generated for
 characterization of DNA-VLPs. It contains the following subfolders: 
 */AGE.* This sub-folder contains raw, uncropped electrophoresis data
 (.tif). Each file is labeled with the experiment it shows. */SDS-PAGE.*
 This subfolder shows raw, uncropped SDS-PAGE data (.tif) for eOD-GT8
 characterization. */TEM images raw.* This folder contains raw transmission
 electron microscopy micrographs exported as .tiff files. The data is
 organized into sub-folders denoting each particle being characterized and
 magnification used. (e.g. d40-30mer/67K). #### **File:
 Fig1_S3_cryoEM.tar.gz** **Title/Description:** **Class averages and
 constituent particles for d40-30mer 2D classifications.** This file is a
 compressed tar.gz containing raw CryoEM micrographs (.mrc) and class
 averages (.mrc, .star) for d40-30mer DNA-VLPs shown in Fig. 1 and Fig. S3.
 Class averages were generated in Relion from 14,000 hand-picked particles.
 #### **File: Fig_S7.zip** **Title/Description:** **Comparison of GC
 responses induced by d40-30mer with common adjuvants.** This file contains
 data used to assess DNA-VLPs with different adjuvants. The contains flow
 cytometry files (.fcs) and AGE gels (.tif). The folder contains the
 following-subfolders:  */AGE*. This sub-folder contains the raw, uncropped
 electrophoresis data (.tif) showing d40-30mer VLPs incubated with
 different adjuvants. (From left to right: 1kb Plus ladder, no adjuvant,
 SMNP, Alum, AddaVax, As01b, CpG). */FSC files*. This folder contains raw
 flow cytometry data for characterization of lymph nodes after immunization
 with DNA-VLPs and different adjuvants. The data is further organized into
 sub-folder based on the adjuvant used (e.g., FCS-Alum, FCS-AddaVax., etc.)
 and folder containing single color controls used for compensation. Each
 mouse replicate is labeled 01-04 for n=4 mice/group. #### **File:
 Fig_S8.zip** **Title/Description:** **Characterization of serum stability
 and vaccine responses in DNase I KO mice.** This file contains AGE gels
 (.tif) and flow cytometry (.fcs) used to characterize DNA-VLP stability
 and immune responses in DNase-deficient mice. This folder contains the
 following sub-folders:  */AGE*. This folder contains raw, uncropped
 electrophoresis data (.tif) characterizing DNA stability in sera and
 plasma from DNase I KO or WT mice. This folder is further sub-divided in
 sub-folders for each experiment (/DNase I KO genotyping, /ssDNA or dsDNA
 stability in WT vs KO; /VLP stability in WT vs KO). */FSC-DNAse KO_p60mer
 immunization.* This folder contains .fcs files used to evaluate GC
 responses in DNase I KO or WT mice after immunization with p60mer
 nanoparticles. It is sub-divided into folders including single color
 controls for compensation (/FCS-compensation) and folders for each mouse
 genotype (/FCS-Dnase KO, /FCS-WT). Each mouse replicate is labeled 01-04
 for n=4 mice/group #### **File: Fig_S9.zip**
 **Title/Description: Polylysine-PEG coating on d40-30mer dampens GC
 responses.** This file contains AGE gels (.tif), flow cytometry (.fcs),
 and confocal microscopy data (.tif) used to evaluate the role of
 polylysine-PEG in DNA-VLP trafficking and GC responses. The folder
 contains the following subfolders:  */AGE.* This folder contains raw,
 uncropped electrophoresis data showing incubation of DNA-VLPs (with or
 without polylysine-PEG treatment) in mouse serum for 0, 2, or 48 hrs. 
 Files are labeled according to treatment (with or without polylysine-PEG).
 */FCS-RAW macrophages.* This folder contains raw flow cytometry data for
 analysis of DNA-VLP uptake in the RAW264.7 cell line. Each FCS file is
 labeled according to treatment (with or without polylysine-PEG) or labeled
 as compensation control. Replicates 1-3 represent staining of 3 different
 wells.  */FCS-GC analysis.* This folder contains raw flow cytometry data
 for analysis of GC responses in mice vaccinated with DNA-VLP with or
 without polylysine-PEG. This data is organized into folders based on
 treatment (/FCS-d40-30mer, /FCS-d40-30mer polyKPEG5k) or compensation
 (/FCS-compensation).  */LN microscopy.* This folder contains .tif images
 of LN sections immunized with d40-30mer or d40-30mer with polylysine-PEG
 (images labeled accordingly). Channel 1 is CD35+FDC staining, channel 2 is
 CD169+SSMs, and channel is fluorescent antigen signal. These images were
 shown for qualitative purposes. #### **File: Fig_S11.zip**
 **Title/Description:** **Whole cleared lymph node images.** This file
 contains confocal microscopy data (8-bit .tif) showing maximum projections
 of whole lymph nodes. Images are labeled according to experimental group.
 ## Code/software All .tif files and .mrc can be accessed using open source
 Fiji/ImageJ software
 ([https://imagej.net/software/fiji/](https://imagej.net/software/fiji/)).
 2D class averages were generated using RELION (v3.0.8) (open source). 
 FlowJo v10 software was used to access and analyze .FCS files containing
 raw flow cytometry data.