Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

Abstract

Efficient antigen delivery to B cells and dendritic cells (DC) is critical for enhancing vaccine immunogenicity. Here, we develop a dimeric vaccine strategy by fusing antigens to the N-terminal of heat shock protein GP96. This platform generates compact, nanoscale particles that fully exposed antigenic sites. We validate the vaccine strategy using the SARS-CoV-2 receptor-binding domain (RBD) antigen in a viral challenge model with hACE2 mice and the human papillomavirus (HPV) E7 protein in a HeLa xenograft model with nude mice. The GP96 moiety directly bound its receptor, LRP1, thereby enhancing antigen accumulation on follicular DCs and prolonging lymph node retention, ultimately amplifying germinal center B cell responses. Furthermore, GP96-LRP1 interaction on DCs promotes antigen endocytosis, underpinning epitope presentation and robust cross-conserved T cell activation. Consequently, this design induces potent, durable humoral immunity, cross-conserved T cell responses, and pulmonary mucosal immunity, underscoring its promise as a versatile and effective vaccination strategy.

Fig. 1. Structure-guided design and critical structure characterization of antigen-GP96-fusion strategy: RBD-GP96-Fusion as an instance.

A Large-scale analysis of the key binding regions of antigens with GP96 using AlphaFold3 (n = 22). B The possible structural pattern diagram of the combination of RBD and GP96 modeled by AlphaFold3. C The schematic representation of RBD-GP96-Fusion (monomer). RBDs (prototype strain) are truncated at the C-terminal residue G545 and connected to GP96 protein N-terminal residue D22 to construct the fusion protein (SP, signal peptide). The structure of the fusion protein was predicted based on AlphaFold3 (yellow RBM motif, hACE2 binding domain). D, E Molecular dynamics of GP96-dimer and RBD-GP96-dimer predicted by AlphaFold2-multimer were performed using AMBER. D The RMSD was analyzed. The red line represents RBD-GP96-dimer and the black line represents GP96-dimer. E The last frame of the molecular dynamics operation of the dimer conformations is captured for display. F Analytical gel filtration of the RBD-GP96-Fusion protein was performed with Superdex 200 Increase 10/300 GL. The resulting 280-nm absorbance curve is shown. The reduced SDS-PAGE migration profile of the pooled sample is shown. The data are representative of two independent experiments with similar results. G Ultracentrifugation sedimentation profiles of RBD-GP96-Fusion. H Transmission electron microscopy of RBD-GP96-Fusion and GP96 protein. The red frames represent typical transmission electron microscopy (TEM) structures. Below is a 3D cartoon sketch of the RBD-GP96-Fusion (surface, cone). I Analysis of GP96 and RBD-GP96-Fusion dimerization models.

Fig. 2. Biological stability and immunological migration characteristics of RBD-GP96-Fusion as a dominant vaccine.

A Representative BIAcore diagrams of RBD and RBD-GP96-Fusion bound to hACE2 protein (baculovirus expression). The K_D value was calculated using the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. B The complex structure of hACE2 (PDB: 1R42) is docked onto RBD-GP96-Fusion dimer by ZDOCK, showing the complete exposure of dual RBMs. Two RBD protomers are shown as surface and colored in hot pink and cyan, respectively. Two hACE2 proteins are shown as cartoon and colored in yellow. C The difference in thermal aggregation degree between RBD, GP96 and RBD-GP96-Fusion proteins was analyzed using the PR.NT.48 instrument. D The SDS-PAGE migration profiles of RBD, GP96 and RBD-GP96-Fusion hydrolyzed by trypsin at different pH were analyzed to determine the difference in anti-enzymatic ability. E Representative in vivo fluorescein image of 6- to 8-week-old female BALB/c mice on days 0, 7, and 30 after dorsal vaccination with RBD (with aluminum hydroxide adjuvant), GP96 and RBD-GP96-Fusion vaccines. In (C, E), n = 3 samples per group were analyzed. The data are representative of two independent experiments with similar results. Geometric mean ± geometric SD are shown. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, *p ≤ 0.05, ****p ≤ 0.0001.

Fig. 3. RBD-GP96-Fusion vaccines elicit robust IgG titers and strong antigen-specific GC B cell responses.

Mice (n = 6 per group) were intramuscularly immunized with SARS-CoV-2 RBD vaccines (prototype or XBB.1.5 strain). A Schematic of RBD-GP96-Fusion induced humoral immunity. B–G Serum analyses at indicated time points. B RBD-specific IgG kinetics at day 7 post-immunization and 6 months after the 3^rd dose. C, D S1-specific and cross-reactive RBD IgG against variants at day 7 post-3^rd dose. E Serum ADCC activity. F, G Neutralization titers (NT50) against SARS-CoV-2 variants at day 7 post-3^rd dose. H Immunization and challenge timeline. I Lung viral RNA by reverse transcription quantitative PCR (RT-qPCR) at day 3 post-infection. J H&E staining of lung tissues (scale bars: 400/100 µm). K, L Bone marrow RBD-specific and XBB.1.5-RBD-specific IgG⁺ ASCs by ELISPOT at 6 months. M, N scRNA-seq of Cy5⁺ cells in draining LNs: t-SNE visualization of B cell clusters (M) and absolute numbers/percentages of subsets (N). O–R Inguinal LN analysis at day 7 post-1^st dose. O Flow cytometry of GC B cells and RBD-specific GC B cells. P, Q Frequencies of GC B cells and RBD-specific GC B cells. R Frequency of RBD-specific CCR6⁺ MBC precursors in LZ. S IgG1⁺ and IgG2a/2b⁺ MBCs frequencies at 6 months. Data represent two independent experiments. Geometric mean ± SD shown; individual points represent mice. L.O.D. indicated (dotted line). Statistical analysis by one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 4. RBD-GP96-Fusion vaccines generate alternative antigen retention mechanisms in GCs via GP96 and FDCs.

Mice were immunized i.m. with SARS-CoV-2 RBD vaccines. Inguinal LNs were collected 7 days post-1^st immunization. A Galaxy plots of monocyte subsets. B Absolute numbers and percentages of each cell type (n = 3). C Frequency of FDCs and RBD⁺/RBD-GP96-Fusion⁺ FDC cells (n = 6). D Flow cytometry analysis of LRP1 and TLRs expression on FDCs (isotype: PE Rat IgG2a, κ). E–G Frequency of RBD⁺/RBD-GP96-Fusion⁺ FDCs after blocking with indicated mAbs. H, I RBD-specific Ab titers at day 14 post-immunization with FcR/LRP1 blocking. J–L IP and Western blot of LRP1-GP96-antigen-BCR complex in FDCs from immunized (J, L) or non-immunized (K) mice. (J) FDC cell sorting was subjected to IP with RBD mAb and Western blotting using RBD and LRP1 mAbs. K FDC cell sorting was incubated with the recombinant RBD protein, RBD protein with GP96 and RBD-GP96-Fusion protein. The incubated FDCs were subjected to IP with RBD mAb and Western blotting by LRP1 mAb. L Lymph node cells were directly subjected to IP by Protein A/G agarose and Western blotting with LRP1, RBD and GP96 mAbs, respectively. M–O GP96-stimulated FDCs enhance GC B cell interactions in LRP1-dependent manner. M FDCs from inguinal LNs were treated with GP96, RBD-GP96-Fusion, and with or without α-LRP1 or isotype control antibody. Hoechst-labeled GC B cells were then added at a 1:1 ratio. Time-lapse images were captured every 5 minutes for 1–2 h to analyze cell interactions and motility. (N) B cell tracking and interaction time quantification (n = 20). O Percentage of B cells with long (> 10 min) vs short (0–10 min) interactions (n = 20). P Frequency of Cy5⁺ FDC or Cy5⁺ GCB cells. Q ZDOCK model of RBD-GP96-Fusion bound to CD91-α receptor. R Schematic of antigen acquisition by FDCs. Data are from n = 6 mice/group unless specified, representative of two experiments. Geometric mean ± SD shown; individual points represent mice. Statistics: one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 5. RBD-GP96-Fusion vaccines induce broad and long-lasting T cell responses by enhancing the targeting of DC cells.

BALB/c or HLA-A2 transgenic mice were immunized with indicated vaccines. A t-SNE plot of T cell subpopulations. B Absolute numbers and percentages of each cell type (n = 3). C Cell-cell contact analysis between CD8⁺ effector and DC cells by CellPhoneDB. D Schematic of cellular immune responses induced by RBD-GP96-Fusion. E, F Spleen and G–K BALF collected at day 14 post-3^rd immunization; L Spleen at 6 months post-3^rd immunization. E, F ICS assay of splenic CD8⁺ T cells producing IFN-γ upon epitope stimulation. G–I An ICS assay was performed to assess the ability of BALFs to secrete IFN-γ following stimulation with cocktail or dominant CD8⁺ epitope S_535-543. G Representative flow plots of BALF CD8⁺IFN-γ⁺ cells. H Frequencies of epitope-specific CD8⁺IFN-γ⁺ cells in BALF. I CD11a and CXCR6 expression on BALF CD8⁺IFN-γ⁺ cells. J, K Frequencies of Trm (J) and α4β7⁺ (K) cells in BALF CD4⁺/CD8⁺ T cells. L AIM⁺ (CD137⁺OX40⁺) CD4⁺ and AIM⁺ (CD137⁺CD69⁺) CD8⁺ T cells in splenocytes after RBD peptide stimulation. M MFI of RBD-TRITC on BMDCs after incubation with indicated proteins. N The kinetics of MFI as (M) over time. O Confocal microscopy of BMDCs showing nucleus (DAPI), LRP1 (FITC), and RBD (TRITC). P Percentage of ROI with LRP1-RBD colocalization. Q Schematic of RBD-GP96-Fusion mediated antigen cross-presentation via CD91/LRP1. Data from n = 6 mice/group (E-L) or n = 6 fields (M–P), representative of two experiments. Geometric mean ± SD shown. Statistics by one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 6. Design and assessment of L1-GP96-Fusion as a vaccine against HPV by fusion strategy.

Groups of 6- to 8-week-old female BALB/c or HLA-A2 transgenic mice (n = 6) were i.m. immunized with HPV-18 L1 vaccines adjuvanted with AS04C, AS04D, MF59, or GP96, and L1-GP96-Fusion vaccine. Naïve mice were also included. A A schematic diagram of HPV-18 L1-GP96-Fusion (monomer). The structure of the L1-GP96-Fusion dimer was predicted based on AlphaFold3. B Reduced SDS-PAGE migration profiles of the pooled samples are shown. C Transmission electron microscopy of HPV-18 L1 and L1-GP96-Fusion protein. The red frames represent typical TEM structures. D Immunization schedule of HPV-18 L1 or L1-GP96-Fusion vaccines. E, F Serum was collected at various time points for analysis by ELISA. Kinetics of L1-specific IgG titers are shown (7 days after immunization). G–I Frequency of GC B cells (G), L1-specific GC B cells (H) or L1-specific MBC precursors (I) at 12 days post 3^rd immunization by flow cytometry. J Frequencies of CD8⁺IFN-γ⁺ cells following stimulation with cocktail in the spleen at 12 days post-3^rd immunization are shown by flow cytometry. K–L The frequency of AIM⁺ (CD137⁺OX40⁺) cells (K) among CD4⁺ T cells and the frequency of AIM⁺ (CD137⁺CD69⁺) cells (L) among CD8⁺ T cells were analyzed (12 days post 3^rd immunization) by flow cytometry. M Representative analysis of various vaccines immune group-mediated killing of MS751 cell line (infected with HPV-18) by flow cytometry. N The frequency of killing percentage at different cell ratios was analyzed as (M). O Spearman correlation of L1-specific CD8⁺IFN-γ⁺ cells frequency and killing percentage. In (E–O), n = 6 mice per group were analyzed. The data are representative of two independent experiments with similar results. The geometric mean ± geometric SD is shown, and each data point represents an individual mouse or field. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 7. Design and evaluation the clearance effect of E7-GP96-Fusion protein against HPV⁺ tumors.

Groups of 6- to 8-week-old female HLA-A2 transgenic mice (n = 6) were i.m. immunized with HPV-18 E7 vaccines adjuvanted with MF59, GP96, and E7-GP96-Fusion vaccine. Naïve mice were also included. A–D Spleens were collected 14 days after 3^rd immunization and stimulated with cocktail for detection. A Representative flow cytometry contour plots of CD8⁺IFN-γ⁺/Granzyme B⁺/Perforin⁺ cells. B The frequency of (A) was analyzed. C Representative flow cytometry contour plots of CD4⁺IFN-γ⁺/Granzyme B⁺/Perforin⁺ cells. D The frequency of (C) was analyzed. E–J Analysis of the clearance effect of CD8⁺ T cell immune response induced by E7-GP96-Fusion in an HPV⁺ tumor challenge model. E Schedule of animal experiments for T cell transfer strategies in HeLa-bearing mice. F Tumor growth curves of HeLa tumor-bearing mice receiving intravenous transfer of different formulations every 3 days, for four immunotherapy sessions. G Photographs and tumor weight H of the collected tumor tissues on day 30. I Representative flow cytometry contour plots of CD8⁺ T cells among tumor-infiltrating lymphocytes (TIL) on day 30. J Frequency, absolute count, and infiltration density of CD8⁺ T cells as shown in (I) was analyzed. In (A–J), n = 6 mice per group were analyzed. The data are representative of two independent experiments with similar results. The geometric mean ± geometric SD is shown, and each data point represents an individual mouse or field. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.