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

Domain: biology
Published: 2025-12-17
Editorial outcome: minor_revision

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.

Introduction
Effective vaccines against continuously emerging and mutating viruses such as SARS-CoV-2 and influenza are challenging to design, and existing vaccines have limited efficiency1,2. The primary concerns are the limited cross-protection and transient nature of the antibody response elicited by the vaccines3. Additionally, while T cells are pivotal in viral clearance and confer long-lasting protection, current vaccination strategies have encountered difficulties in mounting effective T cell responses4. The lack of robust antiviral T cell immune response also limits existing vaccine utility against chronic viral infections such as HPV. Novel vaccination approaches are being actively investigated, which include the utilization of diverse adjuvants, nanoparticle-based antigen delivery systems, artificial intelligence (AI) aided vaccine design, the advancement of mRNA-based or viral vector vaccines, and the engineering of vaccines capable of inducing a more extensive and enduring B cell and T cell response5–9.

Adjuvants strengthen and prolong the immune response by facilitating antigen depot formation, prolonging antigen exposures and release, stimulating innate immunity, and enhancing antigen presentation10,11. The emergence of SARS-CoV-2 variants has complicated the efficacy of current vaccines; however, long-term persistence of neutralizing antibody responses and the diversity and cross-reactivity of T cell responses may serve to ameliorate the clinical impact of these variants12–14. Therefore, vaccine development must prioritize strategies to maintain long-term antibody response as well as the induction of sustained, cross-protective T cell responses through revolutionizing adjuvant development.

Heat shock protein GP96/GRP94, a member of the heat shock protein 90 (HSP90) family, is a pivotal molecular chaperone located within the cellular endoplasmic reticulum (ER). GP96 has emerged as a subject of intense scrutiny in immunological research, with its potential as a vaccine adjuvant to activate T-cell responses being particularly promising15,16. Its ability to complex with a diverse array of MHC class I and class II epitopes, forming GP96-peptide complexes, is crucial for the cross-presentation to CD8+ T cells, a process integral to cellular immune responses17. Additionally, GP96 also stimulates the production of cytokines and chemokines via interaction with CD91 (LRP1) and Toll-like receptors (TLR), thereby inducing the maturation of dendritic cells and macrophages18. The application of GP96 as an adjuvant in split influenza vaccine indicates its potential to elicit protective T cell immune responses against various strains of the influenza virus19. In the realm of tumor immunotherapy, tumor-derived GP96-peptide complexes activate CD8+ T cells by presenting tumor-specific antigens, offering novel and safe strategies for clinical cancer treatment20,21. Despite the notable advancements in GP96 immunological function, the precise form of antigen binding by GP96 remains to be fully elucidated, and its relatively weak ability to promote B-cell responses potentially limits its application in vaccine strategies22.

Given the uncertainty surrounding the binding of large antigens to GP96, we adopt a fusion protein strategy to redesign the antigen-GP96 complex using a structure-based approach23–25. In this study, we construct antigen-GP96 fusion proteins using the RBD of the SARS-CoV-2 spike protein and the L1 and E7 proteins of HPV. Their immunogenicity is compared with that of established adjuvants, including aluminum salts, MF59, AS04C, and AS04D. The results demonstrate that this vaccine strategy induces potent humoral immunity, cross-protective T cell responses, and pulmonary mucosal immunity. These findings establish a novel vaccine platform for developing highly effective and broad-spectrum vaccines against diverse pathogens.

Results
Structure-guided design of a dimeric form of RBD-GP96-Fusion as a superior immunogen
Screenings related to different antigen-GP96 binding modes were first carried out in light of the adjuvant characteristics of GP96. The binding of multiple viral antigens was simulated using AlphaFold3, including SARS-CoV-2 RBD, FluA HA1, HPV L1, HBV HBc, HIV gp120, MPXV B6R and M1R, HFMD CA16 and EV71, and OVA, whose molecular weight is mainly concentrated within the range of 35 to 50 kDa. And the modeling results are corrected for deviations and subjected to orthogonal experiments to ensure their rationality (Supplementary Fig. 1). After large-scale screening and statistical analysis, it was found that these antigens tended to predominantly bind to the N-terminal domain (NTD), and secondly to the middle domain (MD) of GP96 (Fig. 1A, B, and Supplementary Fig. 1, 2A). Therefore, fusing the antigens to the N-terminal of GP96 is more conducive to reducing the spatial hindrance between the antigens and GP96, further ensuring the integrity of the recombinant protein structure and the full exposure of the antigens. Moreover, we analyzed the GP96 binding sites with different subtype antigenic MHC class I/II epitopes that are usually less than 30 aa. The results show that the GP96 dimer uses its lumenal channel formed within its MD and C-terminal domain (CTD) to bind various viral epitope peptides for cross-presentation and induce T cell immune responses (Supplementary Fig. 2B), which is in line with previous reports26.

Unlike epitope peptides, the viral antigens (such as RBD and L1 protein), which are much larger in size, were unable to form stable complexes with GP96 protein (Supplementary Fig. 2C). This offers a valuable reference for our subsequent design of the antigen-GP96-Fusion vaccine strategy. The mature GP96 protein contains a flexible region (aa 22-45) at the N-terminal that could be used as a natural linker to allow RBD to interact with NTD. Additionally, by leveraging the dimerization characteristic of both the C-terminal of GP96 and RBD, a more stable RBD-GP96 fusion protein with dimerization at both the N- and C-terminal will be formed. The RBD is truncated at the C-terminal residue G545 and connected with GP96 protein N-terminal residue D22 to construct fusion protein (Fig. 1C). Molecular dynamics simulation results indicate that the root mean square deviation (RMSD) fluctuations of the RBD-GP96-dimer tend to be more stable compared to the GP96-dimer (Fig. 1D). The flexibility of Chain A and Chain B of GP96 dimer decreased when combined with RBD, particularly for Chain B (Supplementary Fig. 3A). The snapshot of the last frame of the dynamics simulation was then intercepted to represent a more reasonable conformation of the GP96 and RBD-GP96 dimer complex for further analysis. After RBD was coupled to its N-terminal, a large number of hydrogen bonds were formed between RBD Chain A/B and GP96 Chain A/B, further facilitating the stability of the N-terminal of the dimer (Fig. 1E, and Supplementary Fig. 3B). Then recombinant RBD-GP96-Fusion was expressed, and its molecular weight was determined to be 231 kDa through analytical gel filtration and analytical ultracentrifugation, suggesting stable dimer formation (Fig. 1F, G). Transmission electron microscopy observations show that the dimerization of RBD-GP96-Fusion is more compact at both the N- and C-termini compared to GP96 (Fig. 1H). Based on the analysis of the mass spectrometry results regarding the positioning information of disulfide bonds, disulfide bonds are formed mutually between Cys258 (Chain A) - Cys258 (Chain B) of the two RBDs among partial RBD-GP96-Fusion proteins, further facilitating the formation of dimerization conformations: “closed” conformation at the N-terminus through a disulfide bond, and “open” conformation at the N-terminus with no disulfide bonds (Fig. 1I, and Supplementary Fig. 4). Two different twisted models of RBD-GP96-Fusion are consistent with the results of structural predictions.

The surface plasmon resonance (SPR) and ELISA assays indicated that the RBD-GP96-Fusion bound to the SARS-CoV-2 receptor hACE2 with an affinity comparable to the RBD dimer counterpart (Fig. 2A, and Supplementary Fig. 5A). This implies the exposure of the receptor binding motif (RBM), which is in accordance with the results of multiple structural predictions (Fig. 2B, Supplementary Fig. 5B). The aggregation temperatures (Tagg) of GP96 and RBD-GP96-Fusion were significantly higher than that of RBD (Fig. 2C, Supplementary Fig. 6A). Meanwhile, the anti-trypsin enzymatic cleavage ability of GP96 or RBD-GP96-Fusion was significantly enhanced compared to RBD alone (Fig. 2D). Obviously, the enhanced stability of RBD-GP96-Fusion is majorly dependent on the properties of GP96. The results of the in vivo stability study showed that the duration for RBD-GP96-Fusion to exist stably, acting like a sustained-release depot, at the injection site on the backs of mice and subsequently in the inguinal lymph nodes was significantly prolonged compared to that of RBD and GP96 proteins (Fig. 2E). A larger amount of fluorescently labeled RBD was captured by the inguinal lymph nodes (LN) as early as 3 h after the subcutaneous injection of RBD-GP96-Fusion, and the majority of the RBD antigen persisted within the lymph nodes and spleen for at least 30 days.

RBD-GP96-Fusion vaccine promotes the generation of potent neutralizing antibodies via activation of GCB cell response
To evaluate humoral responses, BALB/c mice were immunized three times with RBD-GP96-Fusion or RBD immunogen plus alum or GP96 adjuvant as controls (Fig. 3A). The antibody response to RBD-GP96-Fusion is dose-dependent, and a sufficient high-titer antibody can be produced when immunized with 20 μg of protein (Supplementary Fig. 7A). The inoculation dosage of 20 μg RBD-GP96-Fusion protein was used in the following experiment. The dose of 5 μg of RBD was then used for inoculation in the control groups to maintain the same molar amount of RBD immunogen as in RBD-GP96-Fusion group11. Compared to the RBD with alum or GP96, the RBD-GP96-Fusion of the prototype SARS-CoV-2 induced significantly higher levels of antigen-specific IgG, IgG1, IgG2a, IgG2b and IgM, and no obvious increases in IgA and IgE levels were observed in serum (Fig. 3B, and Supplementary Fig. 7B-E). The mean IgG titer under RBD-GP96-Fusion treatment was up to 63 times higher than that of alum after the 3rd immunization, which persisted for 6 months. In terms of cross-antibody reactions against different SARS-CoV-2 strains of RBD and S1 subunit, RBD-GP96-Fusion immunity was significantly stronger than other treatments (Fig. 3C, D). We found that RBD-GP96-Fusion could induce a strong antibody-dependent cellular cytotoxicity (ADCC) reporter activity in mouse cells (Fig. 3E)27,28.

We then detected the major inflammatory cytokines in the plasma. As seen in Supplementary Fig. 7F, only a modest increase in IFN-γ and TNF levels was observed after the third immunization of RBD-GP96-Fusion relative to alum adjuvant, suggesting that it did not trigger a systemic inflammatory response11. We also monitored the weight change curves of mice throughout the entire vaccination period and did not observe any significant weight loss under RBD-GP96-Fusion treatment (Supplementary Fig. 7G). No other apparent toxicity was found.

In terms of vaccine protection efficacy, all serum samples from the RBD-GP96-Fusion group also exhibited a much higher neutralizing activity against both live prototypic and Omicron BA.1 strains, with approximately a 63-fold and 4-fold increase, respectively, compared to alum (Fig. 3F, and Supplementary Fig. 7H, I). Additionally, neutralizing antibody (NAb) titers demonstrated a stronger correlation with IgG titers (Supplementary Fig. 7J). We further applied this N-terminal fusion strategy to reconstitute the SARS-CoV-2 XBB.1.5 RBD-GP96-Fusion vaccine for immunization (Supplementary Fig. 8A). Similar to the prototypic RBD, the XBB.1.5 RBD-GP96-Fusion generated stronger antigen-specific IgG and NAbs (Fig. 3G, and Supplementary Fig. 8B), and the NAb titers against Omicron BA.1 were enhanced significantly (Supplementary Fig. 8C). After immunization of hACE2 transgenic mice, a nasal SARS-CoV-2 XBB.1.22 challenge test was conducted. The RBD-GP96-Fusion group showed significantly better protective effect than RBD plus alum group, with the lower viral titer in the lungs and no signs of severe bronchopneumonia or interstitial pneumonia in the pathological sections (Fig. 3H–J). Then we compared bone marrow SARS-CoV-2-specific Ab-secreting cells (ASC) elicited by the different vaccines 6 months post-immunization. Consistent with the serum IgG data, mice immunized with prototypic or XBB.1.5 RBD-GP96-Fusion but not RBD plus alum or GP96 generated RBD-specific IgG+ long-lived plasma cells (LLPC) (Fig. 3K, L), suggesting efficient generation of multiple facets of long-term humoral immunity by RBD-GP96-Fusion vaccines. Notably, the RBD-GP96-Fusion barely produces antibodies against its own GP96 (Supplementary Fig. 8D).

We then proceeded to investigate the immune cell repertoire bound by RBD-GP96-Fusion protein via flow cytometry and single-cell RNA sequencing (scRNA-seq), as illustrated in Supplementary Fig. 9A. As shown in Supplementary Fig. 9B, C, RBD-GP96-Fusion protein accumulates significantly more at the draining lymph nodes of the mice. Among Cy5+ cells, we initially grouped the scRNA-seq data into 8 cell clusters, including clusters of B cells, T cells, monocytes, etc., to provide an analytical framework for further in-depth refinement (Supplementary Fig. 9D, E).

Next, we subclustered B cells and obtained 5 subsets according to the expression of canonical B cell markers29–31 (Supplementary Figs. 10A, B). As seen in Fig. 3M-N, compared with the RBD control group, the RBD-GP96 fusion protein was most bound to germinal center (GC) B cells. The enrichment of functional modules of differentially expressed genes (DEG) in the various cell types indicated that the shared upregulation module was predominantly enriched in GC B cells, and was focused on amino acid metabolism as well as formation of immunological synapse with antigen-presenting cells (APC) (Supplementary Figs. 10C, D).

Subsequently, GC B cells from the draining inguinal LNs were assessed 7 days post-immunization by flow cytometry (Fig. 3O). The RBD-GP96-Fusion immunization significantly induced a higher frequency and quantity of GC B cells and antigen-specific GC B cell responses compared to alum or GP96 (Fig. 3P, Q and Supplementary Fig. 11A). The same GC B cell responses were maintained but slightly diminished on day 40 (Supplementary Fig. 11B). To understand the long-term efficacy of antibody levels further, we measured GC memory B-cell (MBC) precursors (as defined in Supplementary Fig. 11C) in the inguinal LNs 7 days post-immunization. We found that a substantial population of RBD-specific MBC precursors was present only upon immunization with RBD-GP96-Fusion vaccines (Fig. 3R, and Supplementary Fig. 11A). MBCs were measured in the spleen of mice 6 months after vaccination. RBD-GP96-Fusion vaccines promoted the generation of class-switched MBCs expressing IgG1 and IgG2a/2b, but no significant difference was observed in RBD specificity7,32 (Fig. 3S, and Supplementary Fig. 11D, E). The immunization with XBB.1.5 RBD-GP96-Fusion is also consistent with the GC responses mentioned above (Supplementary Fig. 11F–H). To better study MBCs and MBC precursors, the formation of MBCs was analyzed 8 weeks after the third immunization, that is, after the GC collapse. At this time, the differences in MBC precursors were no longer significant, and the differences in memory cells were more concentrated on the high-intensity IgG1 and IgG2a/b MBCs. The progress of the promoting kinetics conforms to the above-mentioned GC transformation (Supplementary Fig. 12).

After analyzing the above immune response, it was found that the quantity of RBD-GCB cells shows a strong correlation with IgG and NT50 titer at different immunization stages (Supplementary Fig. 13A–C). Regarding the maintenance of antibodies, the strong correlation between RBD-specific ASCs and long-lasting antibodies in the RBD-GP96-Fusion group also demonstrated its superiority (Supplementary Fig. 13D).

Besides BALB/c mice, induction of humoral response by RBD-GP96-Fusion was also determined in C57BL/6 background mice33. Despite a somewhat weakened humoral response compared to BALB/c mice, RBD-GP96-Fusion induced significantly stronger antibody response and GCB-related reactions than those of alum or GP96 adjuvant control in HLA-A2(Kb)/B6 transgenic mice (Supplementary Fig. 14).

The RBD-GP96-Fusion vaccine generates an alternative antigen retention route via LPR1 on FDCs for continuous activation of GCB cells
Then we subclustered monocyte cells and obtained 6 subsets according to the expression of canonical cell markers34 (Supplementary Fig. 15A, B). There was a significant increase in RBD-GP96-Fusion protein-bound follicular dendritic cells (FDC) compared to RBD control (Fig. 4A, B). The enrichment of functional modules of DEGs indicated the upregulation of cytokine-mediated signaling and regulation of cell-cell adhesion (Supplementary Fig. 15C), suggesting that the RBD-GP96-Fusion protein promotes direct or indirect interactions between FDCs and targeted cells.

FDCs are dynamic antigen libraries in the GCs, providing the first signal for activating GC B cells35–37. T follicular helper (Tfh) cells can provide the second signal for activating GC B cells by secreting corresponding cytokines7. There were no significant differences in the frequency, quantity, and activation of Tfh cells between the RBD-GP96-Fusion group and the groups with RBD with alum or GP96 overall (Supplementary Fig. 16). A relatively higher percentage of IFN-γ+ or IL-4+ Tfh was observed in the RBD-GP96-Fusion group under stimulation with PMA/Ionomycin or RBD peptide pool. FDCs have the unique ability to retain intact antigen for extended periods. They primarily bind antigen-immune complexes onto the cell membrane via surface Fc fragment receptor (FcR) and complement receptor 2/complement receptor 1 (CR2/CR1), and subsequently present them to B cells. FDCs were evaluated in LNs 7 days post-immunization by flow cytometry38,39 (Supplementary Fig. 17A). In the RBD-GP96-Fusion group, the frequency of RBD-GP96-Fusion-specific FDCs exceeded that of RBD-specific FDCs in the RBD with alum group by around 1.3-fold. Furthermore, even after applying FcR blocking inhibitors, the disparity persisted (Fig. 4C, Supplementary Fig. 17B). This indicates that aside from the classical antigen retention pathways involving FcR and CR2, the RBD-GP96-Fusion antigen can be retained on the surface of FDC through other routes.

We postulate that there may exist GP96-related receptors on the surface of FDCs, enabling RBD-GP96-Fusion to anchor the antigen more abundantly on the surface of FDCs via GP9640–42. The results of flow cytometry indicated that both TLRs and LRP1 were expressed on the surface of FDCs (Fig. 4D). Then, FDCs were evaluated in LNs 7 days post-immunization. The combination of FcR blocking, TLR mAb, and LRP1 mAb inhibitors was employed to detect the frequency variations of antigen-specific FDCs. The results revealed that blocking both FcR and LRP1 mostly decreased the binding of the RBD-GP96-Fusion antigen to FDCs, and the combination blocking almost completely inhibited the binding (Fig. 4E–G, and Supplementary Fig. 17C). However, only blocking FcR, not LRP1, significantly decreased the binding of RBD to FDCs. Similar to the binding to FDCs, the results of in vivo immune experiments in mice also demonstrated that LRP1 blocking led to a significant reduction in IgG titers (Fig. 4H, I). After the first immunization, the lymph nodes were extracted and ground, and FDCs were sorted for RBD IP-Western analysis (Fig. 4J, Supplementary Fig. 17D). Likewise, for the unimmunized lymph nodes, FDCs were sorted and incubated with various proteins before undergoing RBD IP-Western analysis (Fig. 4K). The in vivo and in vitro results both indicated the existence of the RBD-GP96-Fusion-LRP1 complex on the surface of FDCs. After one immunization, the lymphocyte suspension was used for Protein A/G beads to directly bind B cells’ BCR and perform IP-Western blot. The results directly proved that B cells and FDCs were bound by the RBD-GP96-Fusion mediated by LRP1 (Fig. 4L).

Next, we assessed the ability of in vitro RBD-GP96-Fusion-stimulated FDCs to prolong the interaction with GC B cells, as measured by time-lapse live-cell microscopy. Figure 4M shows representative FDC-GC B interactions over time. The interaction time between FDCs and GC B cells was significantly enhanced by RBD-GP96-Fusion, and could be blocked by LRP1 antibodies (but not by an isotype control antibody) (Fig. 4N, O). Additionally, after immunization, the frequency of RBD-GP96-Fusion-bound FDCs and GC B cells in the LNs was significantly higher than RBD-bound cells in the alum adjuvant group (Fig. 4P, and Supplementary Fig. 18).

From a structural perspective, AlphaFold3 was utilized to model and analyze LRP1/CD91α. Based on research conducted by Robert J. Binder, we determined that the free GP96 subunit binds to CD91-α through a structural proline-cleavage site to evaluate the feasibility of the vaccine conformation40 (Supplementary Fig. 19). Subsequently, GP96 and RBD-GP96-Fusion were docked with CD91-α for analysis. The results revealed that the docking sites were all situated on the free subunit, suggesting that the docking results merit consideration (Supplementary Fig. 20). At this point, the proposed solution is feasible, as indicated by the fully exposed RBM in RBD (Fig. 4Q). To enhance the structural platform’s subsequent application potential, we analyzed the CD91α-GP96 binding interface via molecular dynamics, and identified the top 10 amino acids within CD91 or GP96 contributing the most to binding energy of CD91α-GP96 (Supplementary Fig. 21, Supplementary Table 1). The most binding-energy-contributing amino acids of GP96 were then mutated to Ala, and WT and mutant GP96 proteins were expressed, purified and characterized (Supplementary Fig. 22A-F). Experiments showed that these mutations within GP96 significantly affected its binding to CD91α (Supplementary Fig. 22G), thus verifying the binding interface between GP96 and CD91α. Based on the above mentioned experiments, a schematic diagram of the mode in which the RBD-GP96-Fusion vaccine is fixed on FDCs in GCs through binding to LRP1/CD91-α, in addition to FcRs and CR2 pathways, to enhance GC B cell activation, is presented as shown in Fig. 4R. By using the presented model that combines the results of Figs. 2D and 2E, we proposed that the RBD-GP96-Fusion strategy can enhance antigen stability, enter B cell follicles more effectively through sinuses, and target FDCs surface LRP1 with GP96 to accumulate more intact antigens for B cells, thereby causing a strong and long-lasting humoral immune response.

RBD-GP96-Fusion vaccines induce broad and long-lasting T cell responses by targeting DCs
Given the superiority of the interaction between CD11c+ DCs and effector T cells with the GP96, we proceeded to subcluster T cells and identified 7 subsets based on the expression of canonical T cell markers (Supplementary Fig. 23A, B). As depicted in Figs. 5A and 5B, there was a notable increase in CD8 effector T cells bound to the RBD-GP96-Fusion protein compared to the RBD control. The enrichment of functional modules of DEGs in the various cell types indicates that the shared upregulation module is predominantly enriched in CD8 effector cells and focuses on leukocyte-mediated cytotoxicity, regulation of cell killing, and regulation of lymphocyte-mediated immunity (Supplementary Fig. 23C, D). Additionally, cell interactions between CD8 effector cells and cDCs are increased in RBD-GP96-Fusion protein-treated mice compared to RBD-control (Fig. 5C), as calculated by CellPhoneDB.

Then cellular immune response was examined in BALB/c and HLA-A2 transgenic mice after immunization (Fig. 5D). The RBD-GP96-Fusion immunization demonstrated higher levels of secreted TNF and IFN-γ by splenocytes, indicating a more prone Th1-type cellular immunity compared to the RBD with alum or GP96 group (Supplementary Fig. 24A). Further design of H-2-Kd and HLA-A2 epitopes was carried out43–45 (Supplementary Table 2). After the third immunization, epitope-specific T cell immune responses were detected by ELISPOT (Supplementary Fig. 24B, C), and the conservation of these epitopes was predicted (Supplementary Fig. 24D). After screening out the highly efficient and conserved epitopes S535-543 and S424-432, intracellular cytokine staining (ICS) assays were conducted (Fig. 5E, F and Supplementary Fig. 25A). The above results showed that the RBD-GP96-Fusion vaccine could induce highly efficient cellular immune responses and had conservation against different virus strains. The results of the ICS assay and the frequencies of activation-induced marker (AIM)+ CD4+ and CD8+ T cells stimulated by S535-543 or SARS-CoV-2 RBD peptide pool after the first immunization showed that the RBD-GP96-Fusion vaccine was significantly more effective than the RBD with alum or GP96 group (Supplementary Fig. 25B-D), suggesting that the RBD-GP96-Fusion vaccine may be able to establish cellular immune protection at an early stage.

Compared to RBD with alum or GP96 group, the proportion of CD8+ T cells secreting IFN-γ in the bronchoalveolar lavage fluid (BALF) of the RBD-GP96-Fusion group after the third immunization sharply increased by 5.3-fold and 8.2-fold respectively (Fig. 5G, H, and Supplementary Fig. 26A), indicating that the vaccine could induce immunodominant epitope-specific CD8+ T cells in the lung and airways43,46. Moreover, the expression of CD11a and CXCR6 in CD8+ T cells of the BALF was significantly enhanced (Fig. 5I, and Supplementary Fig. 26A, B), suggesting that the lung cellular immune response induced by RBD-GP96-Fusion vaccine might be driven by the corresponding chemokines for migration. And to further confirm that the drive originated from the lungs, we detected the lung tissue-resident memory T cells (Trm) and the corresponding integrins43,46–48. The results showed that RBD-GP96-Fusion group had significantly higher levels of CD69+CD103+ Trm of CD4+ and CD8+ T cells, as well as α4β7+ CD4+ and CD8+ T cells than control groups (Fig. 5J, K, and Supplementary Fig. 26C–I). However, the generation of RBD-specific IgA antibodies in the lungs could not be detected (Supplementary Fig. 26J).

To assess the long-term durability of cellular immunity maintenance, the spleen immune responses were examined 6 months after the third immunization. The peptide pool-stimulated AIM+ CD8+ T cell immune response in the RBD-GP96-Fusion group remained significantly higher than the RBD with alum or GP96 group, whereas there was no significant difference in the AIM+ CD4+ T cell immunity49 (Fig. 5L, and Supplementary Fig. 27). Among the main subsets of memory T cells, the stem cell-like memory T cells (Tscm) were significantly higher in the RBD-GP96-Fusion group (Supplementary Fig. 28), indicating that this cell subset is vital for maintaining long-term T cell immunity in this immune group50. After stimulation with the peptide pool, the corresponding antigen-specific activated effector memory T cells (Tem) and IFN-γ+ CD8+ Naïve T/Tem subtypes of memory T cells showed a significant increase (Supplementary Fig. 29). The cellular immune responses elicited by the SARS-CoV-2 XBB.1.5 RBD-GP96-Fusion vaccine constructed with the same strategy align with the trends observed in the above experiments (Supplementary Fig. 30).

The variation of the RBD signal intensity on the cell membrane surface after co-incubation of different antigens with bone marrow dendritic cells (BMDC) can indicate that RBD-GP96-Fusion can be enriched on the surface of BMDCs more quickly and in greater quantities (Fig. 5M, N). The immunofluorescence co-localization signals indicated that the antigen enrichment occurred through the binding of GP96 and CD91/LRP1 (Fig. 5O, P). Subsequently, the cross-presentation pathway after antigen endocytosis was analyzed via immunofluorescence co-localization signals, based on various treatments with endocytosis and autophagy-lysosomal inhibitor BAFA1 and proteasome inhibitor MG132. It was observed that more RBD was delivered to the ER under treatment with RBD-GP96-Fusion than RBD with Alum or GP96, and this process was strongly inhibited by BAFA1 and MG132 (Supplementary Fig. 31, 32). Similar results were observed on the Raw264.7 cell line (Supplementary Fig. 33). The results indicate that after endocytosis of the RBD-GP96-Fusion vaccine, it undergoes hydrolysis in the lysosome or proteasome, and RBD peptides are delivered to the ER for antigen presentation and T cell activation (Fig. 5Q, Supplementary Fig. 34 presents a schematic diagram).

We further conducted flow cytometry validation of major differential immune cell populations bound by the RBD-GP96-Fusion protein, following the process outlined in Supplementary Fig. 9A. The differences in absolute cell counts were consistent with the scRNA-seq conclusion (Supplementary Fig. 35), demonstrating the reliability of the above conclusion.

The HPV vaccine constructed by a fusion strategy induces potent antibodies, cellular immune responses, and virus clearance ability
To validate the universality of this GP96 fusion strategy, another vaccine was designed. For the modification of the HPV vaccine, the HPV-18 L1 is truncated at the C-terminal residue G535 and connected with GP96 protein N-terminal residue D22 to construct fusion protein51. The dimer structure was predicted using AlphaFold3, revealing that the L1 dimer primarily interacts with the NTD domain of GP96 (Fig. 6A, and Supplementary Fig. 36A, B). Additionally, the interaction sites between the L1-GP96-Fusion protein and the CD91α subunit align with those shown in Supplementary Fig. 20A, allowing for proper exposure of the L1 antigen (Supplementary Fig. 36C). The fusion protein was purified and analyzed by gel electrophoresis and TEM (Fig. 6B, C), revealing a stabilized compact conformation with both N- and C-terminal dimerization. The melting temperature (Tm) of L1-GP96-Fusion was significantly higher than that of HPV-L1 (Supplementary Fig. 37).

BALB/c mice were immunized three times, and their spleens and sera from various stages were collected for detection (Fig. 6D). Immunization was performed using 20 μg of L1 protein in combination with widely recognized HPV adjuvants (AS04C, AS04D, and MF59) as positive controls. The L1-GP96-Fusion induced a certain degree of improvement in antibody titer and quantification (Fig. 6E, F). There is no significant patternization difference in the types of antibody reactions and cross-immunity between L1-GP96-Fusion and classical adjuvants, except that L1-GP96-Fusion induced higher IgG2a production (Supplementary Fig. 38A–D), indicating enhanced Th1-type immune response. Corresponding to the antibody response, L1-GP96-Fusion immunization can also elicit a potent GCs response, causing a significant increase in the frequency of L1-specific GC B cells and MBC precursors (Fig. 6G–I). However, no significant differences in L1-specific IgG ASCs could be induced in the short term (Supplementary Fig. 38E).

The ICS assays showed a significantly increased trend in effector CD8+ T cell response following L1-GP96-Fusion immunization, mainly reflected in the differences of CD8+ IFN-γ+ and Granzyme B+ cytokines compared to immunization with L1 with AS04C/D, MF59 or GP96 adjuvant, suggesting a correlation with cytotoxic function (Supplementary Fig. 39, Fig. 6J). Additionally, the trends of AIM+ T enhancement were consistent (Fig. 6K, L). Nevertheless, the results of ELISPOT and the examination of epitope conservation were rather disordered, and the monitoring of memory T cell responses still need to be conducted over a longer period of time52 (Supplementary Fig. 40, and Supplementary Table 3). We further employed HLA-A2 transgenic mice for immunization. The cell cytotoxicity assays using the HLA-A2 cell line MS751 expressing HPV type 18 virus demonstrated that the killing ability of the L1-GP96-Fusion immunization against virus-infected cells was much stronger than that of the classical MF59 adjuvant (Fig. 6M, N, and Supplementary Fig. 41A, B). And this cytotoxic effect is mainly mediated by CD8+ T cells (Supplementary Fig. 41C, D). Considering the correlation between the frequency of CD8+ IFN-γ+ T cells and the observed killing (Fig. 6O), it can be concluded that the T cell immune response primarily contributed to this effect.

We further used HPV E7 antigen to evaluate superior T cell effector responses by GP96-fusion approach, as E6 and E7 were more closely related to the immunotherapy of HPV-driven cancers53,54. We constructed the E7-GP96-Fusion protein (Supplementary Fig. 42A, B). The effector T cell immune responses were detected by the ICS assays with cocktail stimulation 14 days after the third immunization (Supplementary Fig. 42C). E7-GP96-Fusion can induce highly potent effector T cell immune responses, mainly concentrated on the key cytotoxic cytokines CD8+ IFN-γ+, Granzyme B+, and Perforin+ (Fig. 7A–D), which are stronger than those induced by L1-GP96-Fusion. The subcutaneous HeLa xenograft model was used to challenge HPV+ tumors. The effect of E7-GP96-Fusion immunization on tumor growth was investigated by tail vein reinfusion of CD8+ T cells from HLA-A2 transgenic mice immunized with E7-GP96-Fusion or E7 with MF59 or GP96 adjuvant (Supplementary Fig. 42C, D, Fig. 7E). As a result, the smallest tumor volume (Fig. 7F, G), the lowest tumor weight (Fig. 7H), the most CD8+ T cell infiltration were observed in the E7-GP96-Fusion immunization group (Fig. 7I, J).

Methods
All methods in this study were performed in compliance with relevant ethical regulations under a protocol approved by the Institutional Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences.

Mice
Specific pathogen-free (SPF) 6-8-week-old female BALB/c (Stock No. 211), BALB/c nude (Stock No. 401) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HLA-A2.1/Kb (HLA-A2) transgenic mice were maintained in the laboratory62. Specific pathogen-free (SPF) 6-8-week-old female H11-K18-hACE2 transgenic mice were purchased from GemPharmatech Co., Ltd. (Jiangsu, China) (Stock No. T037657). All mice used in this study are in good health and have not been involved in any other experimental procedures. They were housed under SPF conditions in the laboratory animal facilities at Institute of Microbiology, Chinese Academy of Science (IMCAS) and Institute of Laboratory Animal Sciences, Peking Union Medical College. Mice were housed with six companions per cage. All animals were given unrestricted access to water and a standard chow diet and provided with a 12-h light and dark cycle, with a temperature range of 20–25 °C and humidity levels between 40% and 70%. Experimental and control animals were housed separately throughout the study. Euthanasia was performed by the Carbon dioxide (CO2) inhalation or cervical dislocation method. Animal studies were carried out in accordance with the guidelines set forth by the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee under the approved protocol numbers APIMCAS2022128.

Cell culture
Hu94, H7-3, ME180, MS751, SiHa, Hela and RAW264.7 cell lines were obtained from ATCC (Manassas, VA, USA). The Jurkat-mFcγRIII-NFAT-Luc reporter cells (Promega) were donated by the laboratory of Yi Shi (Institute of Microbiology, CAS). The cell lines used in the experiments were all cultured in Roswell Park Memorial Institute 1640 (GibcoBRL, Paisley, UK) with 10% FBS. All culture media contained 100 U/mL penicillin and 100 μg/mL streptomycin. Sf9 (Invitrogen) insect cells were cultured in SIM SF Expression Medium (Sino Biological) at 28 °C.

Protein structure prediction and modeling
AlphaFold3 was used to predict the structure of the monomer of GP96, RBD-GP96-Fusion, LI-GP96-Fusion, and CD91-α. The amino acid sequences of human GP96, SARS-CoV-2 RBD, HPV18 L1, and CD91-α were downloaded from NCBI. And the dimer structures were predicted using the AlphaFold2-Multimer 2.2.4. The ranked_0 structure files of GP96-dimer and RBD-GP96-Fusion dimer were selected for subsequent molecule dynamics analysis. ZDOCK version 3.0.2 was used to predict the complex model of the receptor and ligand via rigid docking. The final results were visualized and analyzed using PyMol 2.5.

Molecule dynamics
The GP96 protein dimer, RBD-GP96-Fusion dimer and CD91-GP96 built using the AlphaFold3 method were simulated separately using full-atom molecular dynamics simulations, which were performed using the AMBER18 software. Before the simulations, the proteins were described using the ff14SB protein force field. Each system was equipped with hydrogen atoms using the LEaP module. A truncated octahedral TIP3P solvent box was added at a distance of 10 Å from the system. Na+/Cl- ions were added to balance the system charge, and the topology and parameter files for the simulation were output. Before the simulations, the system was optimized for energy, including 2500 steps of steepest descent and 2500 steps of conjugate gradient. After the system energy optimization was completed, the system was heated up to 298.15 K at a constant volume and constant heating rate over 200 ps, so that the system temperature was slowly raised from 0 K. Under the condition of maintaining the system temperature at 298.15 K, a 500 ps NVT (constant temperature and constant volume) simulation was conducted to further distribute the solvent molecules evenly in the solvent box. A 500 ps NPT equilibrium simulation was conducted on the entire system, followed by a 100 ns simulation of the two composite systems under NPT periodic boundary conditions. During the simulations, the non-bonded cutoff distance was set to 10 Å. The Particle mesh Ewald (PME) method was used to calculate the long-range electrostatic interactions, while the SHAKE method was employed to limit the hydrogen bond length. The Langevin algorithm was utilized for temperature control, with a collision frequency γ set to 2 ps-1. The system pressure was set to 1 atm, the integration step was 2 fs, and the trajectories were saved every 10 ps for subsequent analysis.

Protein expression and purification
The baculovirus expressed recombinant SARS-CoV-2 RBD, GP96, RBD-GP96-Fusion, HPV18 L1, HPV18 L1-GP96-Fusion, CD91-α, GP96-Mutant, HPV18 E7 and HPV18 E7-GP96-Fusion proteins were expressed with the Bac-to-Bac baculovirus expression system (Invitrogen). The coding sequence for SARS-CoV-2 RBD (S protein residues 319–545, GenBank: PP084047.1), GP96 (residues 22−799, GenBank: NM_003299) and HPV18 L1 (residues 81-535, GenBank: MH057745.1) were codon-optimized and cloned into the baculovirus transfer vector pFastBac1 (Invitrogen). For each construct, gp67 signal peptide sequence was added to the protein N terminus for protein secretion, and a hexa-His tag was added to the C terminus to facilitate further purification processes. Transfection and virus amplification were conducted with Sf9 cells, and the recombinant proteins were produced in Sf9 cells (Invitrogen) for 72 h. The cell supernatants were then collected and soluble proteins were recovered through a 5 mL HisTrap HP column (GE Healthcare). After removal of most of the impurities, the recovered proteins were further purified by gel-filtration chromatography using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) with a running buffer of 20 mM Tris-HCl and 150 mM NaCl (pH 8.0).

Analytical ultracentrifugation
Sedimentation velocity experiments were carried out on two samples (SARS-CoV-2 RBD-GP96-Fusion and HPV18 L1-GP96-Fusion) using the Proteome Lab XL-I analytical ultracentrifuge (Beckman Colter, Brea, CA). A volume of 380 μL of protein sample (A280=0.6 − 0.8) and 400 μL of matching buffer (20 mM Tris, 150 mM NaCl, pH 8.0) were injected into appropriate channels of 12 mm double sector aluminum epoxy cells with sapphire windows. Solutions were centrifuged at 39,000 rpm at 20 °C in an An-60 Ti rotor for 8 h. Scans were collected at 280 nm, with 3 minutes interval between each scan. Data were analyzed using the continuous sedimentation coefficient distribution c(s) model in SEDFIT software.

Transmission electron microscopy structural analysis
Heavy metal salts (such as uranyl formate, uranyl acetate dihydrate, and phosphotungstic acid) were utilized as staining agents for negative staining of proteins for sample preparation. Subsequently, a transmission electron microscope JEM-1400 was employed for the preliminary structural analysis of the samples. Through concentration gradient screening, the optimal resolution concentrations of GP96, RBD-GP96-Fusion, L1, and L1-GP96-Fusion were 2.5 μg/mL, 5 μg/mL, 100 μg/mL, and 5 μg/mL, respectively.

Surface Plasmon Resonance
The Surface Plasmon Resonance (SPR) assays were carried out at 25 °C using a BIAcore 3000 machine with CM5 chips (GE Healthcare). All proteins for SPR assays were exchanged to PBST buffer (10 mM Na2HPO4; 2 mM KH2PO4, pH 7.4; 137 mM NaCl; 2.7 mM KCl; 0.005% Tween 20). The RBD-monomer and RBD-GP96-Fusion of SARS-CoV-2 were immobilized onto CM5 chips in pairs, respectively, at ~1000 response units (RUs). Gradient concentrations of hACE2 (ranging from 3.125 nM to 800 nM for SARS-CoV-2 RBD and RBD-GP96-Fusion) were then flowed over the chip surface at 30 μL/min, and the real-time response was recorded. After each cycle, the sensor surface was regenerated using 7 μL of 10 mM NaOH. The binding kinetics were analyzed using 1:1 binding model with the software BIAevaluation Version 4.1 (GE Healthcare).

Protein thermal stability analysis by nanoDSF
Thermal stability analysis was performed using a PR.NT.48 nanoDSF instrument (NanoTemper Technologies). Purified SARS-CoV-2 RBD, GP96, RBD-GP96-Fusion, HPV-L1 and L1-GP96-Fusion proteins (0.5–1.0 mg/mL in PBS, pH 7.4) were loaded into silica capillaries (10 μL/sample). Samples were heated from 20 °C to 95 °C at 1 °C/min. Melting temperature (Tm) was determined by monitoring the intrinsic tryptophan/tyrosine fluorescence ratio (F350/F330) during unfolding. Aggregation onset temperature (Tagg) was simultaneously measured via back-reflected light scattering at 650 nm. Data were analyzed using PR.Control software (v3.4.0), with Tm and Tagg defined as the inflection points of their respective derivative curves. Experiments were performed in triplicate and results were reported as mean ± SD.

Protein binding assay
The binding of gp96 was quantified by ELISA assay. Briefly, Nunc Maxisorp flat-bottom 96-well plates (Invitrogen, 44-2404-21) were coated with the receptor proteins (10 μg/ml). After blocking with 5% skim milk at 37 °C, serial concentrations of purified gp96 ranging from 0 to 100 μg/ml were added to each well in 100 μl of binding buffer (20 mM HEPES (pH 7.2), 20 mM NaCl, 2 mM MgCl2 and 100 mM KCl) to allow binding for 1.5 h. Afterwards, the plates were incubated with anti-gp96 antibody and the HRP-conjugated secondary antibody. The substrate TMB (3,3’,5,5’-Tetramethylbenzidine) was used for detection. The reaction was measured at 450 nm.

In vivo imaging study
RBD, GP96, or RBD-GP96-Fusion proteins were independently conjugated with R-Cy5 using Lightning-Link R-Cy5 (Abcam, ab188288) kits, following the manufacturer’s instructions. Subcutaneous immunization of mice on the back was conducted with labeled proteins of equal fluorescence intensity and the same molar quantity, and images were recorded with the IVIS Imaging System (Xenogen) 15 min after the injection. Subsequently, fluorescence imaging graphs were captured at various time points in accordance with different studies. The bioluminescence images were analyzed using Living Image software (Xenogen).

Immunization
RBD was diluted in phosphate buffered saline (PBS, Corning, 21-031-CV) and combined in a 1:1 ratio with aluminum hydroxide (Bioss, C07-01013) or with 15 μg recombinant GP96 adjuvant in each dose. RBD-GP96-Fusion was diluted in PBS prior to inoculation. The mixture of HPV18 L1 protein and multiple adjuvants is carried out in the same manner as described above. Each mouse was immunized with 5 μg of RBD protein and 20 μg of RBD-GP96-Fusion protein (of equivalent molar amounts). The immunization strategy for L1 protein was the same as above. Vaccines were injected into the gastrocnemius muscle using a 0.5 mL 28G x 1/2” insulin syringe (BD Biosciences, 329461). For booster immunizations, the same dose of the respective vaccine was injected into the same site as the primary immunization.

Sample collection and processing
After immunization, mice were anesthetized with isoflurane and blood was collected from the orbital sinus using capillary tubes without anticoagulant for serum separation at the time of blood collection. Inguinal and popliteal LNs, spleens, and both tibias and femurs were harvested and placed in cold complete Dulbecco’s Modified Eagle’s Medium [(DMEM, Corning, T10014CV) containing 10% heat-inactivated Fetal Bovine Serum (FBS, Corning, 35-015), 1% Glutamax (Gibco, 35050-061) and 1% Penicillin/Streptomycin (Gibco, 5070063)]. All organs were kept on ice and immediately processed after collection.

LNs and spleen processing: Organs were homogenized with a syringe plunger and filtered through a 40 mm cell strainer on ice. Red blood cells (RBCs) of spleens were lysed using ACK lysing buffer (Lonza, 10-548E) for 5-8 minutes on ice, and the reaction was stopped with cold PBS.

Bone Marrow (BM) processing: BM was flushed from both femurs and tibias from each mouse using a 1 mL 25G x 5/8” syringe (BD Biosciences, 30962). RBCs were lysed as described above.

Cells from all tissues were resuspended in ice cold complete DMEM and immediately used for counting, culture, or staining.

Following collection, blood was centrifuged at 14,000g (maximum speed) for 30 minutes, 4 °C. Serum was recovered and stored at −20 °C for neutralization assays and ELISAs.

ELISA
Nunc Maxisorp flat-bottom 96 well plates (Invitrogen, 44-2404-21) were coated with 2 μg/mL SARS-CoV-2 RBD protein or HPV18 L1 in bicarbonate buffer ON at 4 °C. Plates were washed four times with wash buffer [0.05% Tween-20 (Sigma Aldrich, P1379) in PBS] and blocked with 2% bovine serum albumin (BSA, Fisher Bioreagents, Lot 170763A) in PBS for 1 h at RT. Serum samples were serially diluted in blocking buffer and incubated for 2 h at RT followed by five washes. HRP-conjugated IgG1 (Southern Biotech, 1144-05), IgG2a (Southern Biotech, 1155-05), IgG2b (Southern Biotech, 1186-05), IgG (Southern Biotech, 6412-05), IgA (proteintech, SA00012-7), IgE (Invitrogen, SA5-10263), IgM (Thermo Fisher, 046820) were diluted 1:5,000 in blocking buffer and incubated for 1 h, then washed seven times. Plates were developed with Pierce TMB Substrate (Thermo Scientific, 34021), and the reaction was stopped with 2N sulfuric acid. Absorbance was measured at 450 nm using a SpectraMax microplate reader.

RBD-endpoint antibody titers were calculated as reciprocal dilutions giving OD signals > average of blanks plus three times the standard deviation using GraphPad Prism. An arbitrary value of 1 was assigned to the samples with OD values below the limit of detection for which it was not possible to interpolate the titer.

Antibody-dependent cell-mediated cytotoxicity reporter bioassay
To evaluate antibody-dependent cell-mediated cytotoxicity (ADCC), Jurkat-mFcγRIII-NFAT-Luc reporter cells (Promega, M1211) were used as ADCC bioassay effector cells, and 293FT cells with stable expression of SARS-CoV-2 spike protein were used as ADCC bioassay target cells.

For all ADCC assays, target cells (4.25×103 per well) were incubated with effector cells (7.5×104 per well) and serial dilutions of serum samples were performed at 37 °C for 16 h. After incubation, the relative light unit (RLU) was detected according to the instruction provided by PerkinElmer (Waltham, MA). Fold of induction (FI) was calculated as follows: FI = RLU (induced − background) / RLU (negative serum control − background).

Live SARS-CoV-2 neutralization assay
The live virus neutralization assay was conducted in a BSL-3 facility. Briefly, sera from immunized mice were 2-fold (SARS-CoV-2 neutralization assay) serially diluted and mixed with the same volume of SARS-CoV-2 (100 TCID50), incubated at 37 °C. Thereafter, 100 mL virus-serum mixture was transferred to pre-plated Vero cells in 96-well plates. Inoculated plates were incubated at 37 °C for an additional 72 h, following which the CPE of the virus was observed microscopically at 40-fold magnification. The neutralization titers were defined as the reciprocal of serum dilution required for 50% neutralization of viral infection.

Mice immunizing and viruses challenging
For SARS-CoV-2 challenge experiments, nine hACE2 transgenic mice (8-10-week-old female mice) in each group were intramuscularly immunized in a prime-boost regime. Phosphate-buffered saline (PBS) was employed in the negative control group. Two weeks post the third immunization, mice were infected with 5×105 TCID50 of SARS-CoV-2 XBB.1.22 via the intranasal infection. All mice were euthanized on the 3rd day following the challenge. Lung tissues were harvested for virus load detection (six mice per group) and pathological examination (three mice per group).

Histopathology analysis
Three mice in each group were euthanized on the 3rd day after infection according to standard procedures. Lung samples from challenged mice were collected and immobilized in 10% neutral buffer formaldehyde and embedded in paraffin wax. Tissue sections were treated with hematoxylin and eosin (H&E) and analyzed microscopically.

Fluorescent probe generation
RBD or L1 proteins used for flow cytometry experiments were independently conjugated to both R-APC/Cy7 and R-PE/Cy5 using Lightning-Link R-APC/Cy7 (Abcam, ab102859) and Lightning-Link R-PE/Cy5 (Abcam, ab102893) kits, following manufacturer’s instructions.

ICS and immunophenotyping by flow cytometry
For the detection of cytokine secretion, the final concentration of the stimulated target protein was 20 μg/mL, the final concentration of the polypeptide or peptide library was 20 μg/mL, and the concentration of the Cocktail was used as per the instructions (BioLegend, 423303). The cells were stimulated in 96-well plates for 24 h, with 2×106 cells plated in each well. The Brefeldin A (Invitrogen, 00-4506-51) was added before sample collection, 4−6 h in advance.

All staining steps were carried out at 4 °C in Cell Staining Buffer (BioLegend, 420201). All the steps of cell fixation and membrane disruption are carried out using eBioscience Fixation/Permeabilization Diluent (eBioscience, 00-5223-56). The flow cytometry protocols for various types of immune cells are as follows. Among them, the antigen-APC-Cy7+ antigen-PE-Cy5+ gating strategy was ultimately used for antigen-specific cells.

GCB: CD19+Fas+GL7+
MBC Precursors: CD19+Fas+GL7+CXCR4-CD86+(LZ)
MBCs: CD19+B220+IgD-Fas-CD38+IgG1+/IgG2a/2b+
FDCs: CD31-podoplanin+CD16/32+CD21/35+
Tfh: CD4+B220-CD44hiCD62L-CXCR5+PD-1+IL-21+/IFN-γ+/IL-4+
AIM+ CD4+ T: CD3+CD4+CD137+OX40+
AIM+ CD8+ T: CD3+CD8+CD137+CD69+

ELISPOT
A detailed protocol was previously published8. Briefly, multi Screen HTS IP filter plates of 0.45 μm (Millipore Sigma, MSIPS4W10) were coated with 2.5 μg/mL SARS-CoV-2 RBD or HPV18 L1 in bicarbonate buffer (35 mM NaHCO3 and 15 mM Na2CO3) ON at 4 °C. Plates were washed three times with PBS and blocked with complete DMEM for at least 1 h at 37 °C. Single cell suspensions of BM cells were serially diluted in complete DMEM with halving dilutions starting at one million cells. Following ON incubation at 37 °C and 5% CO2, plates were washed three times with 0.05% Tween-20 in PBS. Membranes were incubated with anti-IgG-HRP (Jackson ImmunoResearch, 115-035-003) detection antibody diluted in complete DMEM for 2 h at RT. Plates were washed three times with 0.05% Tween-20 in PBS, and spots corresponding to antigen-specific antibody-secreting cells (ASC) were developed using BD ELISPOT AEC Substrate Set (551951). Membranes were dried overnight and counted using a CTL Immunospot analyzer (Shaker Heights, OH).

Inhibitor blocking
For the prepared suspension of lymph node cells, in every 2×106 cells FCR blocking (Miltenyi Biotec, 130-092-575) was used at a ratio of 1:10, or TLR1 (Abcam, ab37068), TLR2 (Abcam, ab209216), TLR4 (Abcam, ab22048), and LRP1 (Abcam, ab92544) mAb were used at a ratio of 1:200 for blocking and inhibition. The blocking process was carried out at room temperature for 1 hour. Subsequently, various flow cytometry staining studies were conducted.

Western blot and co-immunoprecipitation assay
In the early stage of the in vivo experiment, mice were immunized with a normal immune dose. Seven days after the first immunization, inguinal and axillary lymph nodes of the mice were collected, and FDC cells were sorted using flow cytometry. In the early stage of the in vitro experiment, mice were not immunized, and FDC cells were directly sorted from the lymph nodes of the limbs. The cells were incubated with 5 μg RBD, 5 μg RBD+15 μg GP96, and 20 μg RBD-GP96-Fusion at 4 °C for 4 h respectively. Subsequently, the cells were lysed with RIPA (Beyotime, P0013B), and the cell lysate was incubated with RBD mAb (1:500, Abclonal, A23771) at a 1:100 ratio at 4 °C overnight. Protein A/G agarose was added and incubated with shaking at 4 °C for 1−2 h. For the BCR-COIP experiment, the lymph nodes of the limbs were collected from immunized mice. They were then ground to obtain a suspension, and the cells were directly lysed. Protein A/G agarose was added and incubated with shaking at 4 °C for 1−2 h. Subsequently, Western Blot analysis was performed for the above three different treatments. The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292T), and LRP1 mAb (1:200, Abcam, ab92544).

The incubation ratio of the primary antibody, HPV18 L1 mAb (Abcam, ab267404), for the detection of HPV-infected cell lines in the Western Blot assay is 1:500.

Live cell imaging and analysis
Separation and isolation of GCB and FDC cells from the inguinal lymph nodes of immunized mice. LRP1 blocking monoclonal antibody (clone 761704; RnD Systems) or rat IgG2a isotype antibody (clone eBR2a; eBioscience) were added 2 h before imaging at a final concentration of 10 μg/ml. Antibody clones and concentrations were based on previously established protocols. Before imaging, GCB cells were labeled with CellTracker Hoechst dye and added to the FDC culture dishes at a 1:1 FDC:B-cell ratio. Cells were imaged on a Nikon A1 inverted microscope (Nikon) using a 40X objective. Images were captured every 5 min for a total of 1 or 2 h. Videos were analyzed using NIS Elements (Nikon) and ImageJ with Manual Tracking plugin. For interaction time analysis, total contact duration per GC B detected from videos was calculated based on the number of frames in which a GC B came in contact with a FDC.

Single-cell RNA sequencing and data processing
Freshly isolated Cy5+ lymph node cells were sorted to >99% purity using a BD FACS Aria III. For the scRNA-seq analysis of Cy5+ sorted cells, each experimental group (including the RBD-in-Alum control) was performed in three independent biological replicates. For each replicate, draining lymph node cells were collected from six mice that had received the same treatment and pooled prior to sorting. Cy5+ lymph node cells were enriched by FACS for library preparation, and scRNA-seq was performed by Shanghai Biotechnology Corporation. The final library pool was sequenced on the Illumina NovaSeq 6000 instrument using 150-base pair paired-end reads. Raw sequencing data were converted to FASTQ files and aligned to the mouse genome reference sequence (GRCH38). The 10X Genomics Cell Ranger (version 3.0.1) was used to demultiplex samples, process barcodes, and generate a digital gene-cell matrix from this data. The output was then imported into the Seurat (version 3.1.5) R toolkit for quality control and downstream analysis. Cells with less than 200 genes and cells with above 20% mitochondrial genes were removed as low-quality cells. Cells with abnormally high unique molecular identifiers or gene numbers were also identified as putative doublets and removed. The Louvain algorithm was used for the unsupervised computational analysis of scRNA-seq data.

Immunostaining for confocal analysis
A 14-mm confocal dish was placed on a 6-well plate. RAW264.7 or BMDC cells were seeded in the 6-well plate at a density of 2×106 cells per well and incubated for 30 min at 37 °C in a cell culture incubator. For intracellular staining in the subsequent steps, inhibitors BAFA1 or MG132 were used for blocking at a ratio of 1:200 for 2−4 hours before protein incubation. Different experimental groups were incubated with 5 μg of RBD, 5 μg of RBD+15 μg of GP96, and 20 μg of RBD-GP96-Fusion for 1 hour. After washing twice with PBS, cells were fixed with 4% paraformaldehyde for 10 min. For subsequent staining of cell surface markers, cells were washed with PBS, while for intracellular staining, cells were washed with 0.5% PBST. A 4% BSA blocking solution was prepared and cells were blocked in a 37 °C cell culture incubator for 15 min. The primary antibody prepared with the blocking solution was added and incubated at room temperature for 3 h. The secondary antibody prepared with the blocking solution was added after washing and incubated at room temperature for 1 h. Following another round of washing, DAPI and mounting reagent were added for subsequent machine analysis. The staining dilution ratios of the antibodies were as follows: LRP1 mAb 1:200 (Abcam, ab92544), RBD mAb 1:500 (Abclonal, A23771), EEA1 mAb 1:100 (CST, 2411S), Rab7 mAb 1:100 (Abcam, ab126712), LAMP1 mAb 1:100 (CST, 9091S), Calnexin pAb 1:500 (Abcam, ab22595), RBD pAb 1:200 (Sino Biological, 40592-MP01), Anti-mouse-TRITC 1:200 (ORIGENE, ZF-0313), and Anti-rabbit-FITC 1:200 (ORIGENE, ZF-0311).

Z-stack images were generated by Imaris (ver 9.7.2). The resulting images were processed by analyzing colocalization, and the percentage of ROI colocalized for cellular membrane and cytosol were obtained respectively. In each assay, a total of 6 fields were randomly selected and quantified.

In vitro cell killing assay
Splenocytes were isolated from immunized mice on day 12 after the 3rd immunization, and collected as effector cells. The target cells (MS751) were labeled with 2.5 μM 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) dye and washed with PBS. We then seeded CFSE-labeled target cells in 100 μL complete RPMI 1640 medium into 96-well round-bottom plates. Effector cells were added to the CFSE-labeled target cells at varying ratios in 100 μL complete RPMI 1640 medium for 4–6 h at 37 °C. The dead cells were labeled with 7-AAD, and cytotoxicity of splenocytes to tumor cells was detected using flow cytometry.

Trypsin protection assay
10 μM purified RBD, GP96 and RBD-GP96-Fusion were incubated at 37 °C for 15 min. The reaction was acidified with increasing amounts of 0.1 M citrate to achieve a series of pH values from pH 7.4 to 5.0 to induce conformational changes in GP96, and incubated for additional 5 min at 37 °C. The solution was then neutralized with 0.5 M Tris (final pH 7.3). 2 μg of trypsin (TPCK [tolylsulfonylphenylalanylchloromethyl ketone] treated; New England Bio-Labs) were added. Digestion was carried out for 1 h at 20 °C, and terminated by the addition of 5× SDS sample loading buffer and heated at 100 °C for 10 min. The extent of GP96 or RBD-GP96-Fusion digestion was analyzed by SDS-PAGE.

BAL and lung harvest and cell Isolation
For mouse samples, spleens were collected, and tissues processed into single-cell suspension. Leukocytes were isolated from spleen by mechanical dissociation and red blood cells were lysed by lysing solution. BAL was harvested directly from euthanized mice via insertion of a 22-gauge catheter into an incision in the trachea. Hanks’ Balanced Salt Solution (HBSS) was injected into the trachea and aspirated 4 times. Recovered lavage fluid was collected and BAL cells were gathered after centrifugation. To isolate intraparenchymal lung lymphoid cells, the lungs were flushed by 5 ml of prechilled HBSS into the right ventricle. When the color of the lungs changed to white, the lungs were excised avoiding the peritracheal lymph nodes. Lungs were then removed, washed in HBSS and cut into 3-mm pieces, and incubated in Iscove’s Modified Dulbecco’s Medium containing 1 mg/ml collagenase IV (Sigma) for 30 min at 37 °C on a rotary agitator (~60 rpm). Any remaining intact tissue was disrupted by passage through a 21-gauge needle. Tissue fragments and majority of the dead cells were removed by a 250-micrometer mesh screen, and cells were collected after centrifugation.

Induction culture of BMDC cells
BMDCs were obtained as previously described63. Briefly, bone marrow cells were harvested from the femur and tibia of 5–7-week old mice, and red blood cells were depleted using red blood lysis buffer. The bone marrow cell suspensions were cultured in complete RPMI medium containing 10% heat inactivated FBS, 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF), 10 ng/mL interleukin-4 (IL-4), and 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). All cytokines were purchased from PeproTech (Rocky Hill, NJ, USA). After 7 days of culture, nonadherent BMDCs were generated and collected.

Peptides synthesis and epitope prediction
The 8-10-mer epitope was predicted using long peptides from the website NetMHC-4.0 (https://services.healthtech.dtu.dk/services/NetMHC-4.0/). The strong binding threshold and weak binding threshold are 0.5% Rank and 2% Rank, respectively. All peptides were synthesized by GenScript (Nanjing, China) and were more than 95% pure.

Quantitative detection of HPV-L1 mouse antibodies by ELISA
Serum from immunized mice was collected and the levels of HPV L1 antibodies were quantitatively detected using the following ELISA kits: Mouse Anti-Human Papilloma Virus 16 late protein L1 IgG (HPV16 L1) ELISA kit, quantitative, 96 tests (Alpha Diagnostic International, 550-316-PMG); Mouse Anti-Human Papilloma Virus 18 late protein L1 IgG (HPV18 L1) ELISA kit, quantitative, 96 tests (Alpha Diagnostic International, 550-318-PMG); Mouse Anti-Human Papilloma Virus 31 late protein L1 IgG (HPV31 L1) ELISA kit, quantitative, 96 tests (Alpha Diagnostic International, 550-331-PMG); Mouse Anti-Human Papilloma Virus 45 late protein L1 IgG (HPV45 L1) ELISA kit, quantitative, 96 tests (Alpha Diagnostic International, 550-345-PMG).

Animal tumor model and adoptive therapy
The subcutaneous HeLa xenograft model was established by inoculating HeLa cells (2×106 cells) into the right foreleg of female BALB/c nude mice (4-to 5-week-old, 18–22 g). At four days after tumor cell inoculation, the tumor-bearing mice were randomly divided into four groups and immunized intravenously in the tail with 3×106 T cells isolated from the spleens of different immunization treatment groups four times at 3-day intervals. Adoptive T cells were enriched using a CD8+ T cell isolation kit (Stemcell, 19853). Tumor growth was monitored with calipers every other day, and the tumor volume was calculated using the following formula: volume = length×width2×0.5. Mice were monitored daily for general health and signs of distress. In accordance with the guidelines of our Institutional Animal Care and Use Committee (IACUC), the following humane endpoints were strictly adhered to, and any animal reaching one of these criteria was euthanized immediately:

Tumor burden: The tumor volume exceeded 1500 mm³.
Ulceration or necrosis: The tumor showed signs of severe ulceration or necrosis.
Body condition: A loss of >20% of initial body weight.
Behavioral signs: Signs of severe pain, distress, or lethargy that inhibited the animal from accessing food or water.
Physical impairment: The tumor size interfered with locomotion or normal bodily functions.
Euthanasia was performed by cervical dislocation under deep anesthesia induced by isoflurane inhalation, a method approved by our IACUC.

Statistical analysis
KD values for SPR assays were calculated by the software BIAevaluation Version 4.1 (GE Healthcare), using 1:1 binding model. The values shown are mean ± SD of two independent experiments. Details can be found in figures legends.

GraphPad Prism version 8 was used to conduct all statistical analysis. Shapiro-Wilk and Kolmogorov-Smirnov tests were performed to establish the normal distribution of the data. The significance of differences among groups was calculated according to the distribution of the data using unpaired one-way ANOVA with Bonferroni correction or two-tailed Mann-Whitney U tests. Correlations were determined using Spearman’s rank coefficient with a 95% confidence interval. Rare samples that presented technical issues in the flow cytometry staining were excluded from the analysis. The precise number of samples analyzed in each graph is reported in figure legends. The specific p-value results of each statistical analysis are presented in the “source-data” file. Statistical significance was set at the critical values of p<0.05 (*), p<0.01 (**), p<0.001 (***), and p<0.0001 (****).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Cheng et al. evaluate the use of heat shock protein GP96 as a vaccine adjunct, leveraging its ability to form dimeric complexes when antigens are fused to its N-terminus. They propose that the structural stability of the GP96-antigen complex enhances antigen retention, promotes localization to follicular dendritic cells, supports germinal center B cell activation, and improves T cell priming. The study also reports enhanced immune responses via improved cross-presentation to CD8⁺ T cells and increased cytokine and chemokine production through engagement of CD91, LRP1, and TLRs. The platform was evaluated using SARS-CoV-2 and HPV antigens, with comparable immunological effects observed in both models.\nThe authors demonstrate rigor in characterizing the structure of the antigen-GP96 fusion protein using mass spectrometry, electron microscopy, and protein gel analyses. They also conduct an in-depth evaluation of both B and T cell responses, including evidence of GP96–CD91/LRP1 interaction via immunofluorescence and inhibition studies in dendritic cells, as well as western blot analysis of DC–GP96-antigen-Bcell complexes.\nMain Comments:\n• Did the vaccine adjunct induce changes in antibody isotype profiles, particularly IgA and IgE levels in serum?\n• Can the authors evaluate whether the vaccine influences effector functions of antibodies, such as complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)?\n• Please comment on the safety and tolerability of the vaccine strategy. Were there any signs of systemic toxicity, weight loss, or inflammation with repeated dosing? Given the involvement of TLRs, what is the risk of triggering a cytokine storm?\n• It would strengthen the translational potential of this platform to more clearly delineate the binding interface between GP96 and CD91α, possibly through additional structural or mutational studies. This could help anticipate interference or competition in future vaccine designs.\nMinor Comments:\n• Please define acronyms such as SPR and RMSD in the main text when first mentioned (line 115), not only in the figure legends.\n• Figure 3I: The legend should be expanded to clarify which assay was used.\n• Figure S28: Please specify the antibody isotype(s) and the assay used in the legend.\nOverall, this manuscript presents a promising and well-validated vaccine delivery platform. The data are extensive and thoughtfully presented, and the study offers meaningful insights into a novel antigen-adjuvant strategy.", "reviewer_2": "Cheng et al. describe development of effective vaccine design with the fusion expression of antigens with N-terminal domain of heat shock protein gp96. Prioritizing the vaccine strategy that maintain long-term antibody responses as well as induction of cross-protective T cell responses makes this work very relevant and significant to the field of vaccine development. Even though the concept of using N-terminal of gp96 is well established by others in the field, Cheng et al describe novel mechanism of targeting LPR1 on follicular dendritic cells that prolongs the retention time and stimulates GC B cells.\nHowever, there are some flaws is the experiment designs:\n1. there is no data showing the dose response curve for RBD-gp96fusion protein. It is not clear why authors use only 5 ug of RBD protein and 15ug of recombinant gp96 adjuvant in the immunization protocol and compare this to immunizations with 20ug RBD-gp96 fusion protein. Authors should provide dose response curve for RBD-gp96 fusion protein and rational for using 20ug of RBD-gp96 fusion protein\n2. Knowing that BLAB/c mice have stronger humoral immune responses then B6, it would be physiologically relevant to test the RBD-gp96 fusion protein in B6 mice as well. especially bc T cell responses were measured in HLA-A2 (Kb) /B6 mice. It is relevant to measure induced B cell and T cell responses in the same mouse strain.", "reviewer_3": "Cheng and colleagues describe a vaccine modality utilizing GP96 to enhance both cellular and humoral immunity. The authors first utilize SARS-CoV-2 Spike RBD as a model immunogen. They provide data supporting that fusion of Ag to GP96 enhances retention of the complex in DLNs on FDCs. This retention is described as then promoting germinal center reaction resulting in enhanced subsequent humoral immune responses. The authors also provide data supporting that fusion to GP96 enhances T cell responses. They then extend their findings using a model HPV antigen which is a target of B cell immunity for protection, HPV18 L1. Here they show antibody, germinal center, and T cell responses comparable or superior to benchmark adjuvant MF59 in immunization studies. While the authors provide data in support of their conclusions (both in the main and supplementary figures), there are a number of weaknesses in this submitted MS that diminish its impact as these issues allow for assessing the impact of this work to the field and also limit the authors stated conclusions. Overall the MS utilizes interesting tools, but requires additional work and immune demonstrations for its full impact for the field.\nMajor comments:\n• A primary limitation for the goals of the manuscript is the lack of a challenge as part of these studies. This makes the data hard to put in context with others work. Immune responses with out impact are a bit out of context. The authors could use a SARS-CoV-2 challenge to support that their vaccine platform confers superior protection to benchmark controls.\nAdditionally, The use of the HPV L1 as a focus on T cells is a bit surprising. HPV E6 and E7 antigens are more relevant for immune therapy of HPV driven cancer as they are required tumor drivers, while HPV L1 is not well expressed after establishment of infection. It is more a target for humoral protection. T cell to L1 would not be expected to clear infection. As there was no HPV+ tumor challenge included if more work is approached perhaps a more relevant HPV immunogen that could be of impact in tumor control could help to support superior T cell effector responses (esp. CD8+ T cells) in the context of this recombinant protein vaccine approach. L1 is currently in the licensed HPV vaccines that are routinely used for prevention. Another comment is that it would be helpful to have more clarity regarding the induced cytokine-secreting CD8+ T cells as the L1 antigen is a larger protein and the induction of an MHC CD8 T cell response would be more informative with significantly more studies of the potential for clearance and the clearance pathway.\n• In a number of figures, the authors provide scRNAseq data from Cy5+ sorted cells as a tool to examine the cell types that were bound by RBD-GP96. They include an RBD in Alum group as a control. However, this is unusual – what receptors are interreacting with the RBD (whether cell surface or endocytic) in T cells? Or in NK cells?\nHow are they Cy5+? While this is referenced for example in Fig 3J-K, Fig S7D-I, Fig 4A-B, 5A-B – there are no statistics included to support this analysis? How many samples were used? The authors should provide additional statistics to substantiate the conclusions. The authors suggest specific claims about these differences in frequency, and their meaning, however what it would be important to present the relationship to the absolute numbers induced?\n• Fig 3J, Fig S8 – An important question to be resolved is the development of germinal center B cells, and memory B cells, etc. which are described induced at 18 hours post immunization? This kinetic appears fast for a germinal center to be specific for the immunizing antigen, and also the induction of memory B cells. If this is 18 hours post prime, this phenotype observed is likely non-specific, called incorrectly, or the existing GCs are from another source depending on the lymph node taken / route. Howeer, this could be interesting to understand. The authors should provide additional data to clarify and strengthen this observation.\n• The authors administer antigen subcutaneously but report that they get BALF responses? What is the mechanism of this observation? It would be important to include the gating strategy for the readers to better interpret this finding. Additionally, to define these as bona fide mucosal responses, various markers would be important to consider such as as CD69, CD103, as well as possibly mucosal homing markers among others. The authors should more rigorously delineate that these responses are not from blood which potential contamination should be ruled out. This would improve the ability to , interpret this finding.\n• The authors focus on MBCs and MBC precursors but do not study recall responses after the GC collapse. Understanding boosting kinetics in terms of humoral immune responses will be important to support formation of MBCs. This can be studied post prime, and then waiting for complete GC collapse (at least 6-8 weeks post immunization).\n• The significance/purpose of the initial docking study of viral antigens fused to GP96 via AF3 is unclear (Fig 1A, S1A/B/C). Were the docking studies used to justify antigen design? If yes, how so?\n• Were the docking studies carried out with replicates (Fig 1A, Fig 6A, S1A/B/C)? Are these docking modes repeatable? Due to the nonexistence of experimentally solved binding interactions (cryo or x-ray) between viral antigens and GP96, AF3 prediction is biased to internal AF3 folding metrics. How are the determinations validated? How do the authors believe the flexible linker impacts antigen exposure and antigen predictions (can it be compared to protein nanoparticles)?\n• In the case of the Sars-CoV-2 RBD dimerization was enhanced through a disulfide bond between the RBDs using disulfide-engineered RBDs described in citation 6. It is not clear in the manuscript that an engineered RBD is used (Fig. 1I, S Fig. 3). Please clarify and discuss this requirement in additional detail.\n• Structural determination in Fig. 1H shows variability and flexibility of the protein that appears to not match the actual predicted structure. Are the authors able to do a 3D reconstruction of the NSEM data or perform cryo-EM to confirm structural integrity? This would be useful to have.\n• In the Protein stability assays in Fig 2 C/D GP96 control is missing, which is necessary to justify whether the protein stability enhancement of the RBD is due to the GP96-fusion or is based on GP96 alone. There is no corresponding method section for Fig 2C. The thermal aggregation experiment in Fig 2C and the isothermal stability assay in S Fig 5A do not correlate. Can you explain why the experiments differ? Is the isothermal stability more physiologically relevant? Were protein stability assays for HPV L1-GP96 fusion proteins performed?\nMinor comments:\n• There are no changes in Tfh cells described, despite changes in GCB – the authors should explain why they think this this is occurring in the discussion section. The authors do note class switching, which would be derived primarily from cytokine signals from Tfh.\n• How is HPV18 L1 antibody quantification performed in Fig 6F. Looking through the methods section the details of this study appear missing.\n• A limitation in the approach is the requirement to engineer the protein to be fused to GP96. This could result in misfolded protein/lack of dimer might impact efficacy for correct B cell engagement. The authors should discuss how to manage this risk in the discussion.\n• Fig S21 among others. The authors discuss various CD4 and CD8 memory populations. This is in reference to the total frequency, not of cytokine+ cells post re-stim. To make stronger conclusions regarding the presence or absence of these populations (and whether they may be antigen specific), the authors should include tetramer staining or peptide re-stim to further analyze these studies.\n• Fig 6M-N – The authors need to separate out CD8s and CD4s rather than study bulk T cells to make their conclusion that the response is primarily CD8 driven.\n• Protein production was carried out using a baculovirus expression system (insect cells), This system does not lead to post-translational modifications (glycosylation). The Authors do not address the potential structural and immunogenicity impact of glycosylation on its viral antigens, an important aspect for the development of targeted antibody reponses relevant to product."}, "author_response": "Reviewer #1 (Remarks to the Author):\nCheng et al. evaluate the use of heat shock protein GP96 as a vaccine adjunct,\nleveraging its ability to form dimeric complexes when antigens are fused to its N-\nterminus. They propose that the structural stability of the GP96-antigen complex\nenhances antigen retention, promotes localization to follicular dendritic cells, supports\ngerminal center B cell activation, and improves T cell priming. The study also reports\nenhanced immune responses via improved cross-presentation to CD8⁺ T cells and\nincreased cytokine and chemokine production through engagement of CD91, LRP1,\nand TLRs. The platform was evaluated using SARS-CoV-2 and HPV antigens, with\ncomparable immunological effects observed in both models.\nThe authors demonstrate rigor in characterizing the structure of the antigen-GP96\nfusion protein using mass spectrometry, electron microscopy, and protein gel analyses.\nThey also conduct an in-depth evaluation of both B and T cell responses, including\nevidence of GP96–CD91/LRP1 interaction via immunofluorescence and inhibition\nstudies in dendritic cells, as well as western blot analysis of DC–GP96-antigen-Bcell\ncomplexes.\nMain Comments:\n• Did the vaccine adjunct induce changes in antibody isotype profiles, particularly IgA\nand IgE levels in serum?\nResponse: Thank you for this insightful and constructive suggestion. We subsequently\ndetected the levels of different types of antibodies in the mouse serum, especially the\nchanges in IgA and IgE levels. It was observed that following the inoculation of mice\nwith the RBD-GP96-Fusion vaccine, the elevation of antibody types in the serum was\npredominantly concentrated in the IgM and IgG classes, with no significant alterations\nin IgA and IgE levels.\nPlease see lines 168-171: “Compared to the RBD with alum or GP96, the RBD-\nGP96-Fusion of prototype SARS-CoV-2 induced significantly higher levels of antigen-\nspecific IgG, IgG1, IgG2a, IgG2b and IgM, and no obvious increases in IgA and IgE\nlevels were observed in serum (Fig. 3B, Fig. S7B-E).”. And please refer to the added\nFig. S7C-E.\n• Can the authors evaluate whether the vaccine influences effector functions of\nantibodies, such as complement-dependent cytotoxicity (CDC) or antibody-dependent\ncellular cytotoxicity (ADCC)?\nResponse: We thank you for this important suggestion. In the revised manuscript, we\nemployed the Jurkat-mFcγRIII-NFAT-Luc reporter cells to conduct additional assays\non the ADCC effect of mouse serum after 3rd immunization1,2.\nPlease see lines 175-177: “And we found that RBD-GP96-Fusion was capable of\ninducing strong antibody-dependent cellular cytotoxicity (ADCC) reporter activity in\nmouse cells (Fig. 3E).”. And please refer to Fig. 3E.\n• Please comment on the safety and tolerability of the vaccine strategy. Were there any\nsigns of systemic toxicity, weight loss, or inflammation with repeated dosing? Given\nthe involvement of TLRs, what is the risk of triggering a cytokine storm?\nResponse: Thank you for these valuable suggestions. The safety and tolerability of the\nvaccine truly merit significant attention. We assayed the levels of multiple key\ncytokines associated with the development of cytokine storms after repeated\nvaccination, and monitored the weight change curves of mice.\nPlease see lines 178-184: “We then detected the major inflammatory cytokines in the\nplasma. As seen in Fig. S7F, only modest increase in IFN-γ and TNF-α levels was\nobserved after the third immunization of RBD-GP96-Fusion relative to alum adjuvant,\nsuggesting that it did not trigger a systemic inflammatory response. We also monitored\nthe weight change curves of mice during the whole vaccination period, and did not\nobserve any significant weight loss under RBD-GP96-Fusion treatment (Fig. S7G). No\nother obvious toxicity was found.”. And please refer to Fig. S7F, G.\nThe following sentences have been added in the Discussion section. Please see lines\n501-509: “Administration of antigen-GP96 fusion vaccine was well tolerate in mice,\nwith no effect on body weight or other obvious toxicity. RBD-GP96-Fusion only\ninduced modest inflammatory response after repeated immunization (Fig. S7F), which\nis far below the threshold level of triggering cytokine storms3. Similar results were\nobserved in other studies using gp96 as adjuvant4,5. Mechanistically, unlike the specific\nligands of TLRs, such as LPS for TLR4, the binding between GP96 as a chaperon and\nits clients TLRs may occur at a dynamic and transient level. The duration and intensity\nof this interaction are relatively weak, and may not trigger excessive and prolonged\nactivation of TLRs and the downstream pathways.”\n• It would strengthen the translational potential of this platform to more clearly\ndelineate the binding interface between GP96 and CD91α, possibly through additional\nstructural or mutational studies. This could help anticipate interference or competition\nin future vaccine designs.\nResponse: We sincerely appreciate your attention to this platform and the invaluable\nsuggestions. Regarding the binding platform of CD91α and GP96, we performed\nmolecular dynamics simulation on the modeling results. Moreover, mutations were\nintroduced at critical binding sites to investigate the alterations in affinity, enabling us\nto obtain a more profound understanding of the interaction interface between CD91α\nand GP96. Additionally, the importance of the research on this binding interface was\nalso elaborated in our discussion.\nPlease see lines 311-319: “To enhance the structural platform's subsequent\napplication potential, we analyzed the CD91α-GP96 binding interface via molecular\ndynamics, and identified the top 10 amino acids within CD91 or GP96 contributing the\nmost to binding energy of CD91α-GP96 (Fig. S21, Table S1). The most binding-energy-\ncontributing amino acids of GP96 were then mutated to Ala, and WT and mutant GP96\nproteins were expressed, purified and characterized (Fig. S22A-F). Experiments\nshowed that these mutations within GP96 significantly affected its binding to CD91α\n(Fig. S22G), thus verifying the binding interface between GP96 and CD91α.” And\nplease refer to Fig. S21, S22 and Table S1.\nThe following sentences have been added in the Discussion section. Please see lines\n520-523: “To further strengthen the translational capacity of this platform in the\nsubsequent research, we conducted a more in-depth analysis of the binding interface\nbetween CD91 and GP96 (Fig. S21, 22). This could allow to anticipate interference or\ncompetition in future vaccine designs.”.\nMinor Comments:\n• Please define acronyms such as SPR and RMSD in the main text when first mentioned\n(line 115), not only in the figure legends.\nResponse: We thank you for this important suggestion. We have expanded the\ndefinitions of these abbreviations in the main text.\nPlease see lines 121-123: “Molecular dynamics simulation results indicate that the\nroot mean square deviation (RMSD) fluctuations of the RBD-GP96-dimer tend to be\nmore stable compared to the GP96-dimer (Fig. 1D).”.\nPlease see lines 142-144: “The surface plasmon resonance (SPR) and ELISA assay\ndemonstrated RBD-GP96-Fusion bound to SARS-CoV-2 receptor hACE2 with\ncomparable affinity as RBD dimer counterpart (Fig. 2A, Fig. S5A).”.\nPlease see lines 175-177: “And we found that RBD-GP96-Fusion was capable of\ninducing strong antibody-dependent cellular cytotoxicity (ADCC) reporter activity in\nmouse cells (Fig. 3E).”.\nPlease see lines 215-218: “The enrichment of functional modules of differential\nexpressed genes (DEGs) in the various cell types indicated that the shared upregulation\nmodule was predominantly enriched in GCB cells, and was focused on amino acid\nmetabolism as well as formation of immunological synapse with antigen-presenting\ncells (APCs) (Fig. S10C, D)”.\nPlease see lines 352-357: “The results of the ICS assay and the frequencies of\nactivation-induced marker (AIM)+ CD4+ and CD8+T cells stimulated by S or\n535-543\nSARS-CoV-2 RBD peptide pool after the first immunization showed that the RBD-\nGP96-Fusion vaccine was significantly more effective than the RBD with alum or\nGP96 group (Fig. S25B-D), suggesting that the RBD-GP96-Fusion vaccine may be able\nto establish cellular immune protection at an early stage.”.\n• Figure 3I: The legend should be expanded to clarify which assay was used.\nResponse: We appreciate your insightful comment. We have made updates to all the\nassay methods employed in the figure legends.\nPlease see lines 1098-1100: “(B) The complex structure of hACE2 (PDB: 1R42) is\ndocked onto RBD-GP96-Fusion dimer by ZDOCK, showing the complete exposure of\ndual RBMs.”.\nPlease see lines 1165-1166: “(H to I) Evaluation of RBD-specific Ab titers kinetics\nwith different blocking inhibitors using ELISA.”.\nPlease see lines 1189-1190: “Frequency of Cy5+FDC or Cy5+GCB cells in inguinal\nLNs at 7 days post-1st immunization with fluorescent labeling is shown by flow\ncytometry.”.\nPlease see lines 1217-1219: “(J and K) The frequencies of Trm (J) and α4β7+ (K)\ncells among CD4+ T cells or CD8+ T cells in BALFs were analyzed by flow cytometry.”.\nPlease see lines 1220-1225: “The frequency of AIM+ (CD137+OX40+) cells (upper)\namong CD4+ T cells and the frequency of AIM+ (CD137+CD69+) cells (lower) among\nCD8+ T cells were analyzed by flow cytometry. (M) Representative mean fluorescence\nintensity (MFI) of RBD-TRITC on BMDC cell surface following incubation with RBD,\nRBD+GP96, or RBD-GP96-Fusion using flow cytometry.”.\nPlease see lines 1249-1259: “(E and F) Serum was collected at various time points\nfor analysis by ELISA. Kinetics of L1-specific IgG titers are shown (7 days after\nimmunization). (G to I) Frequency of GC B cells (G), L1-specific GC B cells (H) or\nL1-specific MBC precursors (I) at 12 days post 3rd immunization by flow cytometry.\n(J) Frequencies of CD8+IFN-γ+ cells following stimulation with cocktail in the spleen\nat 12 days post-3rd immunization are shown by flow cytometry. (K to L) The frequency\nof AIM+ (CD137+OX40+) cells (K) among CD4+ T cells and the frequency of AIM+\n(CD137+CD69+) cells (L) among CD8+ T cells were analyzed (12 days post 3rd\nimmunization) by flow cytometry. (M) Representative analysis of various vaccines\nimmune group-mediated killing of MS751 cell line (infected with HPV-18) by flow\ncytometry.”.\n• Figure S28: Please specify the antibody isotype(s) and the assay used in the legend.\nResponse: Thank you for this valuable suggestion. We have updated both the isotype\ncontrol antibodies utilized and the associated experimental procedures in the figure\nlegends and supplemental information.\nPlease see lines 1160-1162: “Representative analysis of LRP1 and Toll-like receptor\nexpression on the surface of FDC cells measured by flow cytometry. The isotype control\nantibody employed is PE Rat IgG2a (κ isotype ctrl antibody).”. And please refer to\nfigure legends of Fig. S41A, S42D.\nReferences\n1. Wang M, Gao Y, Shen C, et al. A human monoclonal antibody targeting the\nmonomeric N6 neuraminidase confers protection against avian H5N6 influenza virus\ninfection. Nat Commun. 2024;15(1):8871. Published 2024 Oct 15.\ndoi:10.1038/s41467-024-53301-6\n2. He Q, Mao Q, Peng X, et al. Immunogenicity and protective efficacy of a\nrecombinant protein subunit vaccine and an inactivated vaccine against SARS-CoV-2\nvariants in non-human primates. Signal Transduct Target Ther. 2022;7(1):69. Published\n3. Yang J, Wang W, Chen Z, et al. A vaccine targeting the RBD of the S protein of\nSARS-CoV-2 induces protective immunity. Nature. 2020;586(7830):572-577.\ndoi:10.1038/s41586-020-2599-8\n4. Crane CA, Han SJ, Ahn B, et al. Individual patient-specific immunity against high-\ngrade glioma after vaccination with autologous tumor derived peptides bound to the 96\nKD chaperone protein. Clin Cancer Res. 2013;19(1):205-214. doi:10.1158/1078-\n0432.CCR-11-3358\n5. Testori A, Richards J, Whitman E, et al. Phase III comparison of vitespen, an\nautologous tumor-derived heat shock protein gp96 peptide complex vaccine, with\nphysician's choice of treatment for stage IV melanoma: the C-100-21 Study Group. J\nClin Oncol. 2008;26(6):955-962. doi:10.1200/JCO.2007.11.9941\nReviewer #2 (Remarks to the Author):\nCheng et al. describe development of effective vaccine design with the fusion\nexpression of antigens with N-terminal domain of heat shock protein gp96. Prioritizing\nthe vaccine strategy that maintain long-term antibody responses as well as induction of\ncross-protective T cell responses makes this work very relevant and significant to the\nfield of vaccine development. Even though the concept of using N-terminal of gp96 is\nwell established by others in the field, Cheng et al describe novel mechanism of\ntargeting LPR1 on follicular dendritic cells that prolongs the retention time and\nstimulates GC B cells. However, there are some flaws is the experiment designs:\n1. there is no data showing the dose response curve for RBD-gp96fusion protein. It is\nnot clear why authors use only 5 ug of RBD protein and 15ug of recombinant gp96\nadjuvant in the immunization protocol and compare this to immunizations with 20ug\nRBD-gp96 fusion protein. Authors should provide dose response curve for RBD-gp96\nfusion protein and rational for using 20ug of RBD-gp96 fusion protein\nResponse: We thank you for this important suggestion. Prior to establishing the final\nimmunization dose of RBD-GP96 fusion, we carried out an exploration of the\ncorresponding dose response curve. Subsequently, we replenished the experiments on\nthe dose response curve and determined the dose of 20 μg in sequence. Moreover, by\ncalculating the molecular weight ratio where RBD:RBD-GP96-Fusion ≈ 1:4, in the\ncontrol group 5 μg of RBD was inoculated at the same molar amount. This is also\nconsistent with the findings of Yang et al1.\nPlease see lines 163-168: “The antibody response to RBD-GP96-Fusion is dose-\ndependent, and a sufficient high-titer antibody can be produced when immunized with\n20 μg of protein (Fig. S7A). The inoculation dosage of 20 μg RBD-GP96-Fusion\nprotein was used in the following experiment. The dose of 5 μg of RBD was then used\nfor inoculation in the control groups to maintain the same molar amount of RBD\nimmunogen as in RBD-GP96-Fusion group.”. And please refer to Fig. S7A.\n2. Knowing that BLAB/c mice have stronger humoral immune responses then B6, it\nwould be physiologically relevant to test the RBD-gp96 fusion protein in B6 mice as\nwell. especially bc T cell responses were measured in HLA-A2 (Kb) /B6 mice. It is\nrelevant to measure induced B cell and T cell responses in the same mouse strain.\nResponse: We appreciate your insightful comment. Following your suggestion, we\nsupplemented the corresponding experiments and detected the B cell immune response\nafter vaccinating HLA-A2 (Kb)/B6 mice with RBD-GP96-Fusion. Consistent with your\njudgment, the humoral immune response of BALB/c mice is more robust than that of\nB6 mice2. The B cell immune response was still significantly higher than that of the\nalum or GP96 adjuvant group.\nPlease see lines 243-247: “Besides BALB/c mice, induction of humoral response by\nRBD-GP96-Fusion was also determined in C57BL/6 background mice. Despite of\nsomewhat weakened humoral response compared to BALB/c mice, RBD-GP96-Fusion\ninduced significantly stronger antibody response and GCB-related reactions than those\nof alum or GP96 adjuvant control in HLA-A2(Kb)/B6 transgenic mice (Fig. S14).”.\nAnd please refer to Fig. S14.\nReferences\n1. Yang J, Wang W, Chen Z, et al. A vaccine targeting the RBD of the S protein of\nSARS-CoV-2 induces protective immunity. Nature. 2020;586(7830):572-577.\ndoi:10.1038/s41586-020-2599-8\n2. Chu Y, He Y, Zhai W, et al. CpG adjuvant enhances humoral and cellular immunity\nagainst OVA in different degrees in BALB/c, C57BL/6J, and C57BL/6N mice. Int\nImmunopharmacol. 2024;138:112593. doi:10.1016/j.intimp.2024.112593\nReviewer #3 (Remarks to the Author):\nCheng and colleagues describe a vaccine modality utilizing GP96 to enhance both\ncellular and humoral immunity. The authors first utilize SARS-CoV-2 Spike RBD as a\nmodel immunogen. They provide data supporting that fusion of Ag to GP96 enhances\nretention of the complex in DLNs on FDCs. This retention is described as then\npromoting germinal center reaction resulting in enhanced subsequent humoral immune\nresponses. The authors also provide data supporting that fusion to GP96 enhances T\ncell responses. They then extend their findings using a model HPV antigen which is a\ntarget of B cell immunity for protection, HPV18 L1. Here they show antibody, germinal\ncenter, and T cell responses comparable or superior to benchmark adjuvant MF59 in\nimmunization studies. While the authors provide data in support of their conclusions\n(both in the main and supplementary figures), there are a number of weaknesses in this\nsubmitted MS that diminish its impact as these issues allow for assessing the impact of\nthis work to the field and also limit the authors stated conclusions. Overall the MS\nutilizes interesting tools, but requires additional work and immune demonstrations for\nits full impact for the field.\nMajor comments:\n• A primary limitation for the goals of the manuscript is the lack of a challenge as part\nof these studies. This makes the data hard to put in context with others work. Immune\nresponses with out impact are a bit out of context. The authors could use a SARS-CoV-\n2 challenge to support that their vaccine platform confers superior protection to\nbenchmark controls.\nResponse: Thank you for this thoughtful and constructive comment. In accordance with\nyour suggestions, to assess the protective efficacy of the vaccine, we immunized H11-\nK18-hACE2 transgenic mice and subsequently subjected them to SARS-CoV-2\nchallenge. We evaluated the protective efficacy of the vaccine.\nPlease see lines 194-198: “After immunization of hACE2 transgenic mice, a nasal\nSARS-CoV-2 XBB.1.22 challenge test was conducted. The RBD-GP96-Fusion group\nshowed significantly better protective effect than RBD plus alum group, with the lower\nviral titer in the lungs and no signs of severe bronchopneumonia or interstitial\npneumonia in the pathological sections (Fig. 3H-J).”. And please refer to Fig. 3H-J.\nAdditionally, The use of the HPV L1 as a focus on T cells is a bit surprising. HPV E6\nand E7 antigens are more relevant for immune therapy of HPV driven cancer as they\nare required tumor drivers, while HPV L1 is not well expressed after establishment of\ninfection. It is more a target for humoral protection. T cell to L1 would not be expected\nto clear infection. As there was no HPV+ tumor challenge included if more work is\napproached perhaps a more relevant HPV immunogen that could be of impact in tumor\ncontrol could help to support superior T cell effector responses (esp. CD8+ T cells)\nin the context of this recombinant protein vaccine approach. L1 is currently in the licensed\nHPV vaccines that are routinely used for prevention. Another comment is that it would\nbe helpful to have more clarity regarding the induced cytokine-secreting CD8+ T cells\nas the L1 antigen is a larger protein and the induction of an MHC CD8 T cell response\nwould be more informative with significantly more studies of the potential for clearance\nand the clearance pathway.\nResponse: We sincerely appreciate your invaluable suggestions. In the realm of\nimmunotherapy for HPV driven cancers, E6 and E7 stand as pivotal antigens, serving\nas the essential driving factors for tumor development1. This aspect has been a key focus\nof our subsequent investigations.\nConsequently, in line with your recommendations, we have augmented the\nconstruction of recombinant proteins grounded in the HPV E7 antigen. Additionally,\nwe have carried out HPV+ tumor challenges on BALB/c-nu mice1,2. Particular emphasis\nhas been placed on the CD8+ T cell immune response elicited by the E7-GP96-Fusion\nrecombinant vaccine.\nPlease see lines 446-459: “We further used HPV E7 antigen to evaluate superior T\ncell effector responses by GP96-fusion approach, as E6 and E7 were more closely\nrelated to the immunotherapy of HPV-driven cancers. We constructed the E7-GP96-\nFusion protein (Fig. S42A, B). The effector T cell immune responses were detected by\nthe ICS assays with cocktail stimulation 14 days after the third immunization (Fig.\nS42C). E7-GP96-Fusion can induce a highly potent effector T cell immune responses,\nmainly concentrated on the key cytotoxic cytokines CD8+ IFN-γ+, Granzyme B+,\nand\nPerforin+ (Fig. 7A-D), which are stronger than those induced by L1-GP96-Fusion.\nThe\nsubcutaneous HeLa xenograft model was used to challenge HPV+ tumors. The effect of\nE7-GP96-Fusion immunization on tumor growth was investigated by tail vein\nreinfusion of CD8+ T cells from HLA-A2 transgenic mice immunized with E7-GP96-\nFusion or E7 with MF59 or GP96 adjuvant (Fig. S42C, D, Fig. 7E). As a result, the\nsmallest tumor volume (Fig. 7F, G), the lowest tumor weight (Fig. 7H), the most CD8+\nT cell infiltration were observed in the E7-GP96-Fusion immunization group (Fig. 7I,\nJ).”. And please refer to Fig. 7 and Fig. S42.\nFurthermore, in accordance with your suggestions, we measured the cytokines\nassociated with virus clearance secreted by CD8+ T cells induced by L1-GP96-Fusion.\nNotwithstanding that the intensity of the cytokines related to cytotoxicity induced by\nL1-GP96-Fusion was relatively weaker compared to that of E7-GP96-Fusion, a notable\nelevation was still observed in CD8+ Granzyme B+ cells when compared to the MF59\nadjuvant. This finding is correlated with the subsequent in vitro virus clearance and\ncytotoxicity experiments.\nPlease see lines 430-434: “The ICS assays showed a significantly increased trend in\neffector CD8+ T cell response following L1-GP96-Fusion immunization, mainly\nreflected in the differences of CD8+ IFN-γ+ and Granzyme B+ cytokines compared to\nimmunization with L1 with AS04C/D, MF59 or GP96 adjuvant, suggesting a\ncorrelation with cytotoxic function (Fig. S39, Fig. 6J).”. And please refer to Fig. S39.\n• In a number of figures, the authors provide scRNAseq data from Cy5+ sorted cells as\na tool to examine the cell types that were bound by RBD-GP96. They include an RBD\nin Alum group as a control. However, this is unusual – what receptors are interreacting\nwith the RBD (whether cell surface or endocytic) in T cells? Or in NK cells?\nHow are they Cy5+? While this is referenced for example in Fig 3J-K, Fig S7D-I, Fig\n4A-B, 5A-B – there are no statistics included to support this analysis? How many\nsamples were used? The authors should provide additional statistics to substantiate the\nconclusions. The authors suggest specific claims about these differences in frequency,\nand their meaning, however what it would be important to present the relationship to\nthe absolute numbers induced?\nResponse: We thank you for these insightful comments and for pointing out the need\nfor a more detailed explanation of the Cy5⁺ cell populations, as well as the additional\nstatistical analysis.\nIn our study, Cy5⁺ cells were defined as those bound by fluorescently labeled RBD\n(or RBD-GP96-Fusion). The RBD in Alum group served as a protein control to evaluate\nbackground binding/uptake in the absence of GP96-mediated targeting. The use of alum\nwas intended to mimic a conventional protein vaccine formulation, providing a relevant\ncontrol for immunogen exposure in vivo.\nAnd we agree that T cells and NK cells are not canonical RBD-binding cell types.\nIn\nour data, Cy5 positivity in these populations likely arises from:\n(1) Non-specific uptake via Fc receptors on activated lymphocytes binding RBD–\nantibody complexes.\n(2) Endocytic uptake mediated by scavenger receptors or C-type lectins that have\nbeen reported to bind glycosylated proteins, including coronavirus spike domains.\n(3) Troghocytosis or cell–cell transfer from professional antigen-presenting cells\n(e.g., DCs) that have bound RBD and then transiently transfer membrane fragments to\nlymphocytes.\nWe acknowledge that our original figures (e.g., Fig. 3J–K, Fig. S7D–I, Fig. 4A–B,\n5A–B) did not clearly present statistical comparisons or replicate numbers.\nTherefore, we added a description of the number of replicate samples in the revised\nmain text (Methods section). In single-cell RNA sequencing, we set up three biological\nreplicate samples for each group. The cells of each biological detection sample were\ncollected from a mixture of six mice's lymph nodes. In the end, we obtained a total of\nsix groups of single-cell RNA sequencing data for comprehensive analysis. Its NCBI\nSRA number is PRJNA1195897. Please see lines 890-893: “For the scRNA-seq\nanalysis of Cy5⁺ sorted cells, each experimental group (including the RBD-in-Alum\ncontrol) was performed in three independent biological replicates. For each replicate,\ndraining lymph node cells were collected from six mice that had received the same\ntreatment and pooled prior to sorting.”. And please see lines 1025-1028: “The scRNA-\nseq data generated in this study have been deposited in the National Center for\nBiotechnology Information Sequence Read Archive (NCBI SRA) under accession\nnumber PRJNA1195897. A total of six samples of data were obtained. Source data are\nprovided with this paper.”.\nWe agree that frequencies alone may not fully reflect the magnitude of the response.\nTherefore, we analyzed the frequency and absolute cell number of cell subpopulations\nobtained from single-cell RNA sequencing and conducted statistical analysis. These are\nnow included in the revised figures as paired bar graphs showing frequency (%) and\nabsolute counts (as shown in Fig. 3N, 4B, 5B).\nTo verify the results of scRNA-seq, we relabeled Cy5+ RBD-GP96-Fusion and Cy5+\nRBD, and immunized mice following the immunization protocol in Fig. S9A. Then, we\nperformed flow cytometry to quantitatively analyze the three major differential cell\npopulations, GC B cells, effector CD8+ T cells, and FDC cells (for every 30,000 Cy5+\ncells) listed in this article. The results were consistent with those obtained from scRNA-\nseq.\nPlease see lines 400-403: “We further conducted flow cytometry validation of major\ndifferential immune cell populations bound by RBD-GP96-Fusion protein according to\nthe process in Fig. S9A. The differences in the absolute counts of cells were consistent\nwith the scRNA-seq conclusion (Fig. S35). This demonstrated the reliability of the\nabove conclusion.”. And please refer to Fig. S35.\n• Fig 3J, Fig S8 – An important question to be resolved is the development of germinal\ncenter B cells, and memory B cells, etc. which are described induced at 18 hours post\nimmunization? This kinetic appears fast for a germinal center to be specific for the\nimmunizing antigen, and also the induction of memory B cells. If this is 18 hours post\nprime, this phenotype observed is likely non-specific, called incorrectly, or the existing\nGCs are from another source depending on the lymph node taken / route. Howeer, this\ncould be interesting to understand. The authors should provide additional data to clarify\nand strengthen this observation.\nResponse: We sincerely appreciate your identification of this concern. Our analysis of\nthe cells bound by Cy5+ at the 18-hour time point was primarily aimed at discerning the\ndifferences in the cells bound by RBD and RBD-GP96-Fusion within the lymph nodes\nduring the early stage of the immune response. Consequently, it is inevitable that a\ncertain number of non-specifically bound cells were present.\nRegarding your observation that the binding of memory B cells detected in the\nscRNA-seq might be non-specific, we relabeled Cy5+RBD and Cy5+RBD-GP96-\nFusion. Subsequently, after immunization following the protocol outlined in Fig. S9A,\nwe conducted a quantitative analysis of cell numbers via flow cytometry (per 30,000\nCy5+ cells). The results indicated that the number of bound memory B cells was\nrelatively low, and no significant differences among BSA, RBD plus Alum and RBD-\nGP96-Fusion group were observed (Fig. S35B). Thus, we conclude that these bound\nmemory B cells were non-specifically bound.\nHowever, given that the number of these cells by flow cytometry analysis did not\nalign with the number of memory B cell clusters identified in the scRNA-seq, we re-\nevaluated the memory B cell subsets defined in the scRNA-seq. We then refined and\nre-designated it, according to its top 50 marker genes (Table S4) and previous articles3,4,\nand re-labeled it as the more fitting marginal zone B cell (MZB) clusters in the revised\nFig. S10.\n• The authors administer antigen subcutaneously but report that they get BALF\nresponses? What is the mechanism of this observation? It would be important to include\nthe gating strategy for the readers to better interpret this finding. Additionally, to define\nthese as bona fide mucosal responses, various markers would be important to consider\nsuch as as CD69, CD103, as well as possibly mucosal homing markers among others.\nThe authors should more rigorously delineate that these responses are not from blood\nwhich potential contamination should be ruled out. This would improve the ability to ,\ninterpret this finding.\nResponse: Thank you for your insightful comment regarding the need for deeper\nmechanistic validation. With respect to the mechanism underlying the pulmonary\nimmune response induced by subcutaneous antigen injection, especially the T-cell\nimmune response in bronchoalveolar lavage fluids (BALFs) elicited by antigen-\ncarrying GP96, we can draw references from a study of Eva Fisher et al5. It is widely\nbelieved that antigen-presenting cells at the site of immunization imprint subsequent T-\ncell responses and control the expression of transport molecules. Research has found\nthat CD103+ dendritic cells promote the differentiation of tissue-resident memory T\n(Trm) CD8+ T cells and their migration to peripheral epithelial tissues, including the\nlungs, by initiating CD8+ T cells. Eva Fisher et al. have shown that gp96-Ig\nimmunization increases the frequency of CD11chigh MHC IIhigh CD103+ cells at the\nimmunization site. Moreover, CD103+ dendritic cells are a subtype with major\nmigratory capacity and superior cross-presentation ability. Inducing CD103+\ndendritic\ncells through gp96 represents an ideal vaccination strategy for initiating effective and\nlong-lasting immune responses in epithelial tissues. Additionally, the interaction\nbetween CXCR6 and CXCL16 is crucial for controlling the localization of virus-\nspecific TRM CD8+ T cells in the lungs and maintaining the airway TRM cell pool. Our\nexperiments also demonstrated that RBD-GP96-Fusion can induce an increase in CD8+\nCXCR6 expression in the lungs. All of these may be related to the mechanism of CD8+\nT-cell responses in BALFs.\nAccording to your suggestion, we have supplemented the corresponding gating\nstrategy. For details, please refer to Fig. S26A.\nAnd based on your suggestions, in order to better define these as true mucosal\nresponses, we detected the relevant lung-homing markers and excluded contamination\nfrom the blood as much as possible5-8. Please see lines 366-370: “And to further confirm\nthat the drive originated from the lungs, we detected the lung tissue-resident memory T\ncells (Trm) and the corresponding integrins. The results showed that RBD-GP96-\nFusion group had significantly higher levels of CD69+CD103+ Trm of CD4+ and\nCD8+ T cells, as well as α4β7+ CD4+ and CD8+ T cells than control groups (Fig. 5J, K,\nFig. S26C-I).”. And please refer to Fig. 5J, K, Fig. S26C-I.\nAnd please refer to lines 526-534 in the discussion section: “RBD-GP96-Fusion was\nfound to induce CD8+T cell response in BALF (see Fig. 5 and Fig. S26). Eva Fisher et\nal. have shown that gp96-Ig immunization increases the frequency of CD11chigh MHC\nIIhigh CD103+ cells at the immunization site. Together with the results of current study,\nwe propose that RBD-GP96-Fusion immunization may increase CD103+ DCs\nfrequency at the site of immunization, which promote the differentiation of Trm CD8+\nT cells, and thus facilitate their migration to peripheral epithelial tissues including the\nlungs. Additionally, RBD-GP96-Fusion could enhance CXCR6 expression of CD8+ T\ncells that may control T cell localization in the lungs. These mediate the generation of\nT cell immunity in the lungs.”.\n• The authors focus on MBCs and MBC precursors but do not study recall responses\nafter the GC collapse. Understanding boosting kinetics in terms of humoral immune\nresponses will be important to support formation of MBCs. This can be studied post\nprime, and then waiting for complete GC collapse (at least 6-8 weeks post\nimmunization).\nResponse: Thank you for this insightful and constructive suggestion. The research\nemphasis in this paper was placed on the study of germinal center B cells (GCBs),\nwhich inadvertently led to a certain oversight in the research progress of memory B\ncells (MBCs). In accordance with your suggestions, eight weeks after inoculating\nBALB/c mice with three doses of the vaccine, we re-analyzed the differences in\nmemory B cells and their precursors. By this time, the germinal centers had collapsed,\nand the differences in MBC precursors between RBD-GP96-Fusion and alum or GP96\nwere no longer statistically significant. Consequently, the focus shifted to the\ndifferences in MBCs. Moreover, compared with the MBCs six months after\nimmunization, the RBD-GP96-Fusion immunization group demonstrated a more\npronounced increase in IgG1 MBCs.\nPlease see lines 232-237: “To better study MBCs and MBCs precursors, the\nformation of MBCs was analyzed 8 weeks after the third immunization, that is, after\nthe GC collapse. At this time, the differences in MBCs precursors were no longer\nsignificant, and the differences in memory cells were more concentrated on the high-\nintensity IgG1 and IgG2a/b MBCs. The progress of the promoting kinetics conforms to\nthe above-mentioned GC transformation (Fig. S12).”. And please refer to Fig. S12.\n• The significance/purpose of the initial docking study of viral antigens fused to GP96\nvia AF3 is unclear (Fig 1A, S1A/B/C). Were the docking studies used to justify antigen\ndesign? If yes, how so?\nResponse: Thank you for this thoughtful and constructive comment. Based on the\nresearch conducted by John D. Huck et al.9, GP96, as a molecular chaperone, is capable\nof facilitating the folding and processing of client proteins. Thus, in its native state,\nGP96 may exhibit weak interactions with a variety of viral antigens.\nStructurally, GP96 is classified into three major domains: the N-terminal domain\n(NTD), the middle domain (MD), and the C-terminal domain (CTD). When designing\nantigen-GP96 fusion proteins, a crucial consideration is whether to fuse the antigen at\nthe N-terminus or C-terminus of GP96. This approach aims to optimize the antigen-\nGP96 fusion strategy while preserving the integrity of GP96's structure and function.\nPredicting whether the potential binding sites between various viral antigens and\nGP96 are predominantly located in the NTD or CTD can offer valuable insights for our\nconstruction strategy. By doing so, we can strive to ensure that the relative positions of\nthe antigen and GP96 within the antigen-GP96 fusion protein closely mimic their native\ninteraction state, thereby minimizing the disruption of the fusion protein's structure due\nto steric hindrance effects.\nPlease see lines 99-106: “And the modeling results are corrected for deviations and\nsubjected to orthogonal experiments to ensure their rationality (Fig. S1). After large-\nscale screening and statistical analysis, it was found that these antigens tended to\npredominantly bind to the N-terminal domain (NTD), and secondly to the middle\ndomain (MD) of GP96 (Fig. 1A, B, Fig. S1, S2A). Therefore, fusing the antigens to the\nN-terminal of GP96 is more conducive to reducing the spatial hindrance between the\nantigens and GP96, further ensuring the integrity of the recombinant protein structure\nand the full exposure of the antigens.”. And please refer to Fig. 1A, B, Fig. S1, S2A.\n• Were the docking studies carried out with replicates (Fig 1A, Fig 6A, S1A/B/C)? Are\nthese docking modes repeatable? Due to the nonexistence of experimentally solved\nbinding interactions (cryo or x-ray) between viral antigens and GP96, AF3 prediction\nis biased to internal AF3 folding metrics. How are the determinations validated? How\ndo the authors believe the flexible linker impacts antigen exposure and antigen\npredictions (can it be compared to protein nanoparticles)?\nResponse: Thank you for your valuable suggestions. In accordance with your\nsuggestions, we subsequently selected five distinct seeds for each model and employed\nAlphaFold3 for prediction. We then compared the predicted models and calculated the\nRoot Mean Square Deviation (RMSD) distribution to further mitigate the internal\nbiases within AlphaFold3.\nSubsequently, orthogonal experiments were conducted to further compare the\naforementioned predicted models, thereby further ensuring the reliability of the results.\nIt should be noted that the predicted results merely offer guidance and reference. The\nspecific mode of conjugation and whether the flexible linker impacts antigen exposure\nneed to be experimentally verified, for instance, through Surface Plasmon Resonance\n(SPR) experiments. RBD-GP96-Fusion was shown to equally bind to SARS-CoV-2\nreceptor hACE2 and efficiently induce neutralizing Abs mice, partly validating RBD\nantigen exposure when fused with GP96.\nWhen constructing antigen-GP96 fusion proteins, the flexible linker has the\nadvantage of maintaining the structural integrity of antigen-GP96 to the greatest extent\npossible. Nevertheless, its drawback lies in the relatively high degree of freedom, which\nmay cause some crucial antigenic sites to be shielded by the GP96 structure. Hence, it\nis necessary to conduct further experiments to verify whether this strategy is applicable\nto different antigens.\nIn comparison to nanoparticles, GP96 itself can serve as a dimer backbone. Moreover,\ndue to its inherent characteristics, GP96 can effectively induce T cell immune responses\nthrough antigen presentation pathway.\nPlease see lines 99-101: “And the modeling results are corrected for deviations and\nsubjected to orthogonal experiments to ensure their rationality (Fig. S1).”. And please\nrefer to Fig. S1.\nThe following sentences have been added in the Discussion section. Please see lines\n556-560: “RBD-GP96-Fusion was shown to equally bind to SARS-CoV-2 receptor\nhACE2 and efficiently induce neutralizing Abs mice, partly validating RBD antigen\nexposure when fused with GP96. Nevertheless, experimentally solved binding\ninteractions (cryo-electron microscopy and X-ray crystallography) between viral\nantigens and GP96 will be need to avoid potential biases of AlphaFold3 folding metrics.”\n• In the case of the Sars-CoV-2 RBD dimerization was enhanced through a disulfide\nbond between the RBDs using disulfide-engineered RBDs described in citation 6. It is\nnot clear in the manuscript that an engineered RBD is used (Fig. 1I, S Fig. 3). Please\nclarify and discuss this requirement in additional detail.\nResponse: Thank you for this important suggestion. We sincerely apologize for the\nmisunderstanding caused by the inappropriate description. In this study, we did not\nengineer disulfide bonds for the construction of the RBD-GP96-Fusion protein. The\nresearch findings of Dai L were cited herein to illustrate that during the dimerization of\nthe RBD-GP96-Fusion protein10, while the C-terminus of GP96 plays a dominant role,\nthe dimerization of GP96 brings the two RBDs closer in space. As a result, the two\nRBDs can further contribute to the dimerization process by forming disulfide bonds.\nThe disulfide-bond mapping mass spectrometry results presented in Fig. S4 provide\nevidence for the presence of disulfide bonds at the N-terminal of RBD. It should be\nnoted that GP96 is a dynamic molecule, and not all RBDs will dimerize at the N\nterminal. This is consistent with the different conformations observed in the\ntransmission electron microscopy (TEM) results in Fig. 1H, which also echoes your\nnext question.\nWe have meticulously reviewed and rectified the inappropriate descriptions in the\nmanuscript to ensure greater accuracy and clarity. Please see lines 134-141: “Based on\nthe analysis of the mass spectrometry results regarding the positioning information of\ndisulfide bonds, disulfide bonds are formed mutually between Cys258 (Chain A) -\nCys258 (Chain B) of the two RBDs among partial RBD-GP96-Fusion proteins, further\nfacilitating the formation of dimerization conformations: “closed” conformation at the\nN-terminus through a disulfide bond, and “open” conformation at the N-terminus with\nno disulfide bonds (Fig. 1I, Fig. S4). Two different twisted models of RBD-GP96-\nFusion are consistent with the results of structural predictions.”. And please refer to Fig.\n1H, I, Fig. S4.\n• Structural determination in Fig. 1H shows variability and flexibility of the protein that\nappears to not match the actual predicted structure. Are the authors able to do a 3D\nreconstruction of the NSEM data or perform cryo-EM to confirm structural integrity?\nThis would be useful to have.\nResponse: We sincerely appreciate your invaluable suggestions. In our response to the\nprevious question, we noted that the flexible dimerization of GP96 enables the two\nRBDs to come into close proximity, thereby facilitating partial formation of disulfide\nbonds at the N-terminus of RBD-GP96-Fusion dimer. Consequently, the dimerization\nof RBD-GP96-Fusion gives rise to at least two conformations: “closed” conformation\nat the N-terminus through a disulfide bond, and “open” conformation at the N-terminus\nwith no disulfide bonds. The transmission electron microscopy (TEM) results presented\nin Fig. 1H clearly demonstrate the distinct dimer morphologies.\nRegrettably, due to an oversight on our part, we presented only one modeling\noutcome of RBD-GP96-Fusion, which inadvertently led to misunderstanding. To\nrectify this, we have subsequently updated Fig. 1H to include models of RBD-GP96-\nFusion in various conformations. Please see lines 139-141: “Two different twisted\nmodels of RBD-GP96-Fusion are consistent with the results of structural predictions.”.\nAnd please refer to Fig. 1H.\nIt is important to acknowledge that due to the unavailability of the necessary\nconditions, we were unable to perform further structural analysis of this protein using\ncryo-electron microscopy in the short term. This limitation is indeed a shortcoming of\nour study. To mitigate this, we carried out a series of repeated modeling experiments\n(as shown in Fig. S1) and conducted subsequent comprehensive immunological\nfunctional assays. These efforts were aimed at compensating for this limitation to the\ngreatest extent possible. Furthermore, we recognize the crucial role of cryo-electron\nmicroscopy in studies of structural integrity and vaccine antigenic sites. In future\nresearch endeavors, we will endeavor to utilize cryo-electron microscopy for data\nacquisition regarding these recombinant proteins to the greatest extent possible.\nPlease see lines 558-560: “Nevertheless, experimentally solved binding interactions\n(cryo-electron microscopy and X-ray crystallography) between viral antigens and GP96\nwill be need to avoid potential biases of AlphaFold3 folding metrics.”\n• In the Protein stability assays in Fig 2 C/D GP96 control is missing, which is necessary\nto justify whether the protein stability enhancement of the RBD is due to the GP96-\nfusion or is based on GP96 alone. There is no corresponding method section for Fig 2C.\nThe thermal aggregation experiment in Fig 2C and the isothermal stability assay in S\nFig 5A do not correlate. Can you explain why the experiments differ? Is the isothermal\nstability more physiologically relevant? Were protein stability assays for HPV L1-GP96\nfusion proteins performed?\nResponse: Thank you for your valuable suggestions. We have supplemented the\nexperiments of the GP96 protein in Fig. 2C, D and Fig. S6A. Please see lines 146-150:\n“The aggregation temperatures (Tagg) of GP96 and RBD-GP96-Fusion were\nsignificantly higher than that of RBD (Fig. 2C, Fig. S6A). Meanwhile, the anti-trypsin\nenzymatic cleavage ability of GP96 or RBD-GP96-Fusion was significantly enhanced\ncompared to RBD alone (Fig. 2D). Obviously, the enhanced stability of RBD-GP96-\nFusion is majorly dependent on the properties of GP96.”. And please refer to Fig. 2C,\nD, Fig. S6A.\nWe have supplemented the experimental method of Fig. 2C in the Methods section.\nPlease see lines 685-695: “Protein thermal stability analysis by nanoDSF Thermal\nstability analysis was performed using a PR.NT.48 nanoDSF instrument (NanoTemper\nTechnologies). Purified SARS-CoV-2 RBD, GP96, RBD-GP96-Fusion, HPV-L1 and\nL1-GP96-Fusion proteins (0.5–1.0 mg/mL in PBS, pH 7.4) were loaded into silica\ncapillaries (10 μL/sample). Samples were heated from 20°C to 95°C at 1°C/min.\nMelting temperature (Tm) was determined by monitoring the intrinsic\ntryptophan/tyrosine fluorescence ratio (F350/F330) during unfolding. Aggregation\nonset temperature (Tagg) was simultaneously measured via back-reflected light\nscattering at 650 nm. Data was analyzed using PR.Control software (v3.4.0), with Tm\nand Tagg defined as the inflection points of their respective derivative curves.\nExperiments were performed in triplicate; results are reported as mean ± SD.”.\nRegarding the cause of the inconsistent discrepancies between Tm and Tagg, we\npresent the following analysis. The identical Tm values indicate comparable global\nconformational stability (resistance to unfolding) between GP96 and SARS-CoV-2\nRBD, likely due to similar strengths of core structural elements like hydrophobic\npacking or disulfide bonds. However, the significantly higher Tagg (aggregation onset\ntemperature) of GP96 reflects its superior colloidal stability (resistance to aggregation).\nThis difference arises primarily from distinct surface properties and functional\nadaptations: GP96, a molecular chaperone, possesses a more hydrophilic surface,\nhigher net charge promoting electrostatic repulsion, and intrinsic chaperone activity that\ncan shield its own hydrophobic patches during thermal stress. In contrast, the RBD’s\nfunctional requirement for receptor binding necessitates exposed hydrophobic residues\nand flexible loops within its receptor-binding motif, rendering it more prone to\naggregation upon partial unfolding, despite its overall folded stability being similar to\nGP96.\nIsothermal stability (long-term stability at physiological temperature) is generally\nmore physiologically relevant than thermal stability parameters (Tm/Tagg) because\nbiological systems operate at constant temperatures (e.g., 37°C). While Tm and Tagg\nreflect a protein’s resistance to acute thermal stress, isothermal stability directly\nquantifies time-dependent functional longevity under native conditions—such as\nconformational integrity, activity maintenance, and aggregation kinetics over hours to\ndays. This aligns with physiological demands where proteins must resist gradual\ninactivation by proteolysis, oxidation, or colloidal instability. Although Tm/Tagg serve\nas valuable predictors for structural robustness (e.g., low Tm <45 ° C implies\nvulnerability at 37°C), they do not capture chronic stressors or kinetic decay rates. Thus,\nfor therapeutic efficacy, shelf-life, or in vivo behavior, isothermal stability assays (e.g.,\nmonitoring activity loss/aggregation at 37°C) provide direct functional insights,\nwhereas thermal stability offers rapid, complementary screening.\nAs you suggested, we also assayed the thermal stabilities of the L1, GP96, and L1-\nGP96-Fusion proteins. Please see lines 416-417: “The melting temperature (Tm) of L1-\nGP96-Fusion was significantly higher than that of HPV-L1 (Fig. S37).”. And please\nrefer to Fig. S37.\nThe different impact of gp96 fusion on Tagg and Tm between RBD and L1 may be\ndue to different binding regions of these two antigens with GP96.\nMinor comments:\n• There are no changes in Tfh cells described, despite changes in GCB – the authors\nshould explain why they think this this is occurring in the discussion section. The\nauthors do note class switching, which would be derived primarily from cytokine\nsignals from Tfh.\nResponse: Thank you for this important and insightful comment. We have added the\nexplanation in the main text and Discussion section. Please see lines 260-264: “There\nwere no significant differences in the frequency, quantity and activation of Tfh cells\nbetween the RBD-GP96-Fusion group and the groups with RBD with alum or GP96\noverall (Fig. S16). Relatively higher percentage of IFN-γ+ or IL-4+ Tfh was observed\nin the RBD-GP96-Fusion group under stimulation with PMA/Ionomycine or RBD\npeptide pool.”. And please refer to Fig. S16.\nPlease see lines 540-550: “Although the total number of GC-localized Tfh cells\nremained unchanged across experimental groups, we observed significant functional\npolarization, characterized by divergent secretion of the instructive cytokines IL-4 and\nIFN-γ (Fig. S16D-F) in RBD-GP96-Fusion group. This qualitative shift rather than\nquantitative expansion may provide a mechanistic basis for the differential class-\nswitching patterns in GC B cells. Elevated IL-4⁺ Tfh activity promotes IgG1 switching11,\ncorrelating with enhanced anti-RBD IgG1 titers by RBD-GP96-Fusion immunization.\nIncreased IFN-γ⁺ Tfh drives T-bet-mediated IgG2 switching12,13, aligning with the surge\nin IgG2a antibody observed in RBD-GP96-Fusion group. It is likely that alterations in\nTfh cytokine profiles by RBD-GP96-Fusion can reshape GC outcomes, as IL-4 and\nIFN-γ serve as direct molecular switches for antibody subclass commitment.”.\n• How is HPV18 L1 antibody quantification performed in Fig 6F. Looking through the\nmethods section the details of this study appear missing.\nResponse: Thank you for your valuable suggestions. We have supplemented this\ndetection method in the methods section. Please see lines 987-996: “Quantitative\ndetection of HPV-L1 mouse antibodies by ELISA Serum from immunized mice was\ncollected and the levels of HPV L1 antibodies were quantitatively detected using the\nfollowing ELISA kits: Mouse Anti-Human Papilloma Virus 16 late protein L1 IgG\n(HPV16L1) ELISA kit, quantitative, 96 tests (Alpha Diagnostic International, 550-316-\nPMG); Mouse Anti-Human Papilloma Virus 18 late protein L1 IgG (HPV18L1) ELISA kit,\nquantitative, 96 tests (Alpha Diagnostic International, 550-318-PMG); Mouse Anti-\nHuman Papilloma Virus 31 late protein L1 IgG (HPV31L1) ELISA kit, quantitative, 96\ntests (Alpha Diagnostic International, 550-331-PMG); Mouse Anti-Human Papilloma\nVirus 45 late protein L1 IgG (HPV45L1) ELISA kit, quantitative, 96 tests (Alpha\nDiagnostic International, 550-345-PMG).”.\n• A limitation in the approach is the requirement to engineer the protein to be fused to\nGP96. This could result in misfolded protein/lack of dimer might impact efficacy for\ncorrect B cell engagement. The authors should discuss how to manage this risk in the\ndiscussion.\nResponse: Thank you for your crucial suggestion. We have included the mitigation\nstrategy in the discussion section. Please see lines 560-568: “It should be noted that to\navoid misfolded protein or lack of dimer when designing for different antigens,\nunnecessary flexible regions should be removed to the greatest extent possible, and\nlarge-scale protein structure prediction analysis should be combined. Structure\nvalidation and quality control of GP96 fusion proteins are need to ensure efficient\nengagement with B cells. In addition, the structural issues caused by post-translational\nmodifications (such as glycosylation) of proteins can also be optimized through the\nimprovement of expression systems, for instance, the 293F expression system. Protease\ninhibitors can be incorporated to safeguard against protein degradation.”.\n• Fig S21 among others. The authors discuss various CD4 and CD8 memory\npopulations. This is in reference to the total frequency, not of cytokine+ cells post re-\nstim. To make stronger conclusions regarding the presence or absence of these\npopulations (and whether they may be antigen specific), the authors should include\ntetramer staining or peptide re-stim to further analyze these studies.\nResponse: Thank you for this thoughtful and constructive comment. In response to your\nsuggestions, we have supplemented the relevant experiments. Groups of 6- to 8-week-\nold female BALB/c mice (n=6) were i.m. immunized with SARS-CoV-2 RBD vaccines\nadjuvanted with aluminum hydroxide, or GP96, and RBD-GP96-Fusion vaccine.\n8\nweeks after the 3rd immunization, the RBD peptide pool was used for stimulation to\ndetect antigen-specific activated memory T cells.\nPlease see lines 379-382: “After stimulation with the peptide pool, the corresponding\nantigen-specific activated effector memory T cells (Tem) and IFN-γ+ CD8+ Naïve\nT/Tem subtypes of memory T cells showed a significant increase (Fig. S29).”. And\nplease refer to Fig. S29.\n• Fig 6M-N – The authors need to separate out CD8s and CD4s rather than study bulk\nT cells to make their conclusion that the response is primarily CD8 driven.\nResponse: Thank you for your crucial suggestion. According to your suggestion, we\nisolated CD8+ T and CD4+ T from the immunized mice respectively to conduct the\ncytotoxicity assay. Please see line 442: “And this cytotoxic effect is mainly mediated\nby CD8+ T cells (Fig. S41C, D).”. And please refer to Fig. S41C, D.\n• Protein production was carried out using a baculovirus expression system (insect cells),\nThis system does not lead to post-translational modifications (glycosylation). The\nAuthors do not address the potential structural and immunogenicity impact of\nglycosylation on its viral antigens, an important aspect for the development of targeted\nantibody reponses relevant to product.\nResponse: We sincerely appreciate the highly valuable suggestions you put forward\nregarding the subsequent product development.\nIn the early stage of our research, the protein expression platform was primarily\nbased on the insect expression system. Moving forward, we will gradually establish a\nprotein expression platform using 293F cells. All the above-mentioned proteins will be\nre-expressed through the 293F system. This approach aims to delve into the influence\nof glycosylation on the immunogenicity of these proteins.\nFurthermore, we will conduct in-depth research on the impact of different expression\nsystems on the conformation and function of Antigens-GP96-Fusion, with a particular\nemphasis on glycosylation.\nPlease see lines 565-567: “In addition, the structural issues caused by post-\ntranslational modifications (such as glycosylation) of proteins can also be optimized\nthrough the improvement of expression systems, for instance, the 293F expression\nsystem.”.\nReferences\n1. Xu C, Liu W, Hu Y, Li W, Di W. Bioinspired tumor-homing nanoplatform for co-\ndelivery of paclitaxel and siRNA-E7 to HPV-related cervical malignancies for\nsynergistic therapy. Theranostics. 2020;10(7):3325-3339. Published 2020 Feb 10.\ndoi:10.7150/thno.41228\n2. Qin L, Wang J, Cheng F, et al. GPC3 and PEG10 peptides associated with placental\ngp96 elicit specific T cell immunity against hepatocellular carcinoma. Cancer Immunol\nImmunother. 2023;72(12):4337-4354. doi:10.1007/s00262-023-03569-2\n3. Nguyen HTT, Guevarra RB, Magez S, Radwanska M. Single-cell transcriptome\nprofiling and the use of AID deficient mice reveal that B cell activation combined with\nantibody class switch recombination and somatic hypermutation do not benefit the\ncontrol of experimental trypanosomosis. PLoS Pathog. 2021;17(11):e1010026.\nPublished 2021 Nov 11. doi:10.1371/journal.ppat.1010026\n4. Zhang Y, Shen J, Cheng W, et al. Microbiota-mediated shaping of mouse spleen\nstructure and immune function characterized by scRNA-seq and Stereo-seq. J Genet\nGenomics. 2023;50(9):688-701. doi:10.1016/j.jgg.2023.04.012\n5. Fisher E, Padula L, Podack K, et al. Induction of SARS-CoV-2 Protein S-Specific\nCD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice. Front Immunol.\n2021;11:602254. Published 2021 Jan 26. doi:10.3389/fimmu.2020.602254\n6. Zhuang Z, Lai X, Sun J, et al. Mapping and role of T cell response in SARS-CoV-2-\ninfected mice. J Exp Med. 2021;218(4):e20202187. doi:10.1084/jem.20202187\n7.Meng F, Xing H, Li J, et al. Fc-empowered exosomes with superior epithelial layer\ntransmission and lung distribution ability for pulmonary vaccination. Bioact Mater.\n2024;42:573-586. Published 2024 Sep 12. doi:10.1016/j.bioactmat.2024.08.015\n8.Ruane D, Brane L, Reis BS, et al. Lung dendritic cells induce migration of protective\nT cells to the gastrointestinal tract. J Exp Med. 2013;210(9):1871-1888.\ndoi:10.1084/jem.20122762\n9. Huck JD, Que NL, Hong F, Li Z, Gewirth DT. Structural and Functional Analysis of\nGRP94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a\nPotential Client-Binding Site. Cell Rep. 2017;20(12):2800-2809.\ndoi:10.1016/j.celrep.2017.08.079\n10. Dai L, Zheng T, Xu K, et al. A Universal Design of Betacoronavirus Vaccines\nagainst COVID-19, MERS, and SARS. Cell. 2020;182(3):722-733.e11.\ndoi:10.1016/j.cell.2020.06.035\n11. Kubo M. T follicular helper and TH2 cells in allergic responses. Allergol Int.\n2017;66(3):377-381. doi:10.1016/j.alit.2017.04.006\n12. Feng H, Zhao X, Xie J, et al. Pathogen-associated T follicular helper cell plasticity\nis critical in anti-viral immunity. Sci China Life Sci. 2022;65(6):1075-1090.\ndoi:10.1007/s11427-021-2055-x\n13. Weinstein JS, Laidlaw BJ, Lu Y, et al. STAT4 and T-bet control follicular helper\nT cell development in viral infections. J Exp Med. 2018;215(1):337-355.\ndoi:10.1084/jem.20170457"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have reviewed the revised manuscript and find that the authors have adequately addressed my previous comments. I am satisfied with the revisions and have no further concerns.", "reviewer_2": "Authors addressed all of mine concerns and I recommend this article to be published."}, "author_response": ""}]

Decision letter