DNA origami vaccines program antigen-focused germinal centers

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.

Methods
For complete methods, please see accompanying article (Romanov et. al. 2026.)

DNA-VLP synthesis and characterization

DNA-VLPs were assembled as previously described(53). All designs were generated using DAEDALUS(53) (d40 VLP) or ATHENA(49) (d30 VLP) with staples manually adjusted in Tiamat software(87) 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 MgCl2 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 MgCl2, SYBR Safe, 65V for 150 min) and dynamic light scattering. For antigen conjugation, DNA-VLPs were concentrated to >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 MgCl2, SYBR Safe, 65V for 150 min) and quantified using bicinchoninic acid colorimetric assay.

Negative stain transmission electron microscopy

Uranyl formate staining was performed as previously described(28). 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.

CryoEM imaging

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(88) with a particle box size of 320.

Flow cytometry analysis of GC B and Tfh cells

Mice were euthanized at the indicated time point post-injection by CO2 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.

Lymph node microscopy

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 CO2 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(61). 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.

Single cell BCR sequencing

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+/CD38loGL7hi/IgM-IgG+/eOD++KO- populations. We generated BCR libraries from whole transcriptome amplification (WTA) products produced using the Smart-Seq2 protocol on the FACS isolated B cells(89). 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)(90). These primers were also built with attachments to the Illumina P7 (V region) and P5 (constant region) sequences(90). 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(91), and aligned against the human or mouse IMGT database(92).  Sequencing error correction was performed with MigMAP, a wrapper for IgBlast (https://github.com/mikessh/migmap). Consensus VH and VL/VK 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(93). The phylogenetic trees were constructed using the neighbor-joining method with Poisson Model in the MEGAx(94). The reliability of the tree nodes was tested using the Felsenstein bootstrap method with 500 replicates.

Statistics

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 P values are denoted in the figures. For all figures, NS is not significant (P > 0.05).