Severe Acute Respiratory Syndrome Coronavirus-2 Spike Protein Nanogel as a Pro-Antigen Strategy with Enhanced Protective Immune Responses.

Prevention and intervention methods are urgently needed to curb the global pandemic of coronavirus disease-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Herein, a general pro-antigen strategy for subunit vaccine development based on the reversibly formulated receptor binding domain of SARS-CoV-2 spike protein (S-RBD) is reported. Since the poor lymph node targeting and uptake of S-RBD by antigen-presenting cells prevent effective immune responses, S-RBD protein is formulated into a reversible nanogel (S-RBD-NG), which serves as a pro-antigen with enhanced lymph node targeting and dendritic cell and macrophage accumulation. Synchronized release of S-RBD monomers from the internalized S-RBD-NG pro-antigen triggers more potent immune responses in vivo. In addition, by optimizing the adjuvant used, the potency of S-RBD-NG is further improved, which may provide a generally applicable, safer, and more effective strategy for subunit vaccine development against SARS-CoV-2 as well as other viruses.

Introduction

The outbreak of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection has caused a pandemic of coronavirus disease‐19 (COVID‐19), posing a great threat to human life globally.[ ] Till mid‐June of 2020, more than 9 million individuals were tested positive for COVID‐19, with a death toll over 470 000 worldwide.[ ] Early efforts have focused on finding small‐molecule drugs such as favipiravir, chloroquine, and remdesivir to treat SARS‐CoV‐2 infection.[ ] Meanwhile, researchers have also reported the development of effective neutralizing antibodies using techniques such as single B cell sequencing.[ ] Nevertheless, one of the most promising strategies for COVID‐19 prevention relies on vaccine development. There have already been more than 100 vaccines under development, including whole virus vaccines (attenuated, inactivated, or recombinant virus), subunit vaccines, DNA, and RNA vaccines.[ ] For example, an inactivated SARS‐CoV‐2 whole virus vaccine from China showed efficacy in mice, rats, and monkeys.[ ] Another recombinant adenovirus vaccine clinical trial (NCT04313127) has posted its phase 1 results with neutralizing antibodies and reported specific T cell responses.[ ] Whole virus vaccines are expensive, dangerous during production, and may cause severe vaccine‐related diseases.[ ] Alternatively, using subunit vaccines with virus antigen protein should be a safer, more effective, and economic strategy. Recombinant expression of the antigen in organisms such as E. coli, yeast, or mammalian cells can facilitate the large‐scale production.

The receptor binding domain of SARS‐CoV‐2 spike protein (S‐RBD) has been shown to mediate the entry of the virus into host cells via interacting with human angiotensin converting enzyme 2 (hACE2).[ ] The S‐RBD of SARS‐CoV‐1 has been used as a candidate subunit vaccine to prevent virus entry into cells.[ ] When formulated with adjuvants, SARS‐CoV‐1 RBD can elicit protective immune responses.[ ] Since SARS‐CoV‐2 S‐RBD possesses 80% sequence similarity to SARS‐CoV‐1 RBD with an even higher binding affinity to human ACE2,[ ] it should also be a suitable antigen for subunit vaccine development. Nevertheless, the poor pharmacokinetics and low immunogenicity greatly hindered the use of S‐RBD for subunit vaccine development.[ ] A critical reason for the low immunogenicity of S‐RBD lies in its poor targeting ability to lymph nodes, which is crucial for antigen uptake and processing by dendritic cells (DCs) and macrophages.[ ] As nanoparticle formulations have been shown to enhance cell permeability and potency of anti‐cancer drugs,[ ] we envisioned that formulating S‐RBD into redox‐responsive nanogels may serve as a pro‐antigen with improved lymph node targeting and DC and macrophage accumulation, which can lead to synchronized release of internalized S‐RBD monomers with enhanced protective immune responses. Herein, we report a pro‐antigen strategy based on the reversibly formulated spike protein nanogel (S‐RBD‐NG) for subunit vaccine development against SARS‐CoV‐2 (Scheme  ).

Scheme 1

The design of reversibly formulated SARS‐CoV‐2 S‐RBD protein nanogel (S‐RBD‐NG) as a pro‐antigen strategy for subunit vaccine development for COVID‐19. S‐RBD was formulated with redox‐responsive crosslinkers as a pro‐antigen with enhanced lymph node targeting and antigen presenting cell (APC) accumulation. Synchronized regeneration of S‐RBD monomers from the internalized S‐RBD‐NG pro‐antigen triggered more potent immune responses to neutralize SARS‐CoV‐2.

Results and Discussion

We started by overexpressing and purifying SARS‐CoV‐2 S‐RBD from yeast cells and then verified its integrity via SDS‐PAGE and western blotting analysis (Figure S1, Supporting Information). Since SARS‐CoV‐2 S‐RBD contains two N‐linked glycosylation sites, the observed mass from LC‐MS analysis was heterogeneous but higher than the calculated molecular weight without N‐glycosylation (Figure S2, Supporting Information). We further used enzyme‐linked immunosorbent assay (ELISA) to prove that recombinant human ACE2 can bind to S‐RBD expressed in yeast (Figure S3, Supporting Information). The affinity was measured to be ≈7.65 nM (Figure S4, Supporting Information), which was consistent with the reported value.[ ] Furthermore, the interaction can be competed with an S‐RBD targeting SARS‐CoV‐2 neutralizing nanobody, which indicated a similar binding pattern of the yeast‐expressed S‐RBD (Figure S5, Supporting Information).[ ] We then used amine reactive, redox‐responsive reversible chemical crosslinkers to generate protein nanogels (Figure  ). Two crosslinkers with different spacer groups were synthesized (Figure 1B), which contain an internal disulfide bond that is reduced upon uptake by antigen‐presenting cells (APCs) to disassemble the NGs back to protein monomers. Notably, whereas reduction of crosslinker 1 (CL1) would generate a thiol group on the protein, crosslinker 2 (CL2) would undergo a rearrangement to regenerate the native amine on the protein upon reduction (Figure 1C). S‐RBD protein was treated with both crosslinkers at different equivalents and SDS‐PAGE analysis showed that NGs formed by CL2 exhibited a slightly higher efficiency than formed by CL1 and all NGs could be reduced to monomers (Figure 1D). The crosslinked bands were further collected and subjected to dynamic light scattering (DLS) and transmission electron microscopy (Figure 1E,F). In contrast to the native S‐RBD that had a diameter of ≈2 nm, as measured by DLS (Figure S6, Supporting Information), its average diameter was increased to ≈25 nm upon crosslinking, which confirmed the formation of S‐RBD NGs.

Figure 1

Preparation and characterization of reversible SARS‐CoV‐2 S‐RBD nanogels (S‐RBD‐NG) as pro‐antigens. A) Schematic representation of the preparation of S‐RBD nanogels using amine reactive, redox‐responsive reversible chemical crosslinkers with an internal disulfide bond, and N‐hydroxysuccinimidyl ester at both terminals. B) Structures of the two crosslinkers used in this study, CL1 and CL2. C) Schematic representation of the breaking patterns of the crosslinkers in a reducing environment. D) SDS‐PAGE analysis of the prepared S‐RBD nanogels and their disassembly under reducing conditions. CBB: Commassie brilliant blue staining. Cy5: Cy5 fluorescence. E) Dynamic light scattering analysis of S‐RBD‐NG. F) Transmission electron microscopy image of S‐RBD‐NG. Scale bar: 50 nm.

As the uptake of antigens by APCs (DCs and macrophages) is critical for antigen processing and cross‐presentation, we first examined the internalization of S‐RBD‐NGs by these cells. Confocal microscopy was used to quantify the uptake by DC2.4 or RAW 264.7 cells after incubation with NGs formulated using different S‐RBD/crosslinker ratios (Figure  , Figures S7 and S8, Supporting Information). Compared to the S‐RBD monomer, enhanced cellular uptake of S‐RBD‐NGs prepared with both CL1 and CL2 was observed, and quantitative analysis of the imaging data showed an approximately fourfold enhancement of uptake with 50 equivalents of crosslinkers (Figure 2B,D). Since reduction of CL2 can regenerate native proteins, we used CL2 at a 50:1 ratio with S‐RBD to produce NGs for the following study.

Figure 2

Uptake of S‐RBD or S‐RBD‐NG by DCs and macrophages. Confocal images of A) DC2.4 cells and C) RAW 264.7 cells that internalized S‐RBD and S‐RBD‐NG after incubation for 24 h. Scale bar: 50 µm. Quantitative analysis of the cellular uptake of S‐RBD and S‐RBD‐NG by B) DC2.4 cells and D) RAW 264.7 cells. Data are presented as mean ± SEM. n = 3.

Next, we tested whether S‐RBD‐NG could improve lymph node targeting ability in vivo. Cy5‐labeled S‐RBD or S‐RBD‐NG were administered to C57BL/6N mice via intramuscular injection, and inguinal lymph nodes were collected after 24 h (Figure  ). Ex vivo imaging showed significantly higher accumulation of S‐RBD‐NG in lymph nodes compared to S‐RBD monomers (Figure 3B). Further quantitative analysis showed an ≈3.9‐fold enhanced accumulation of S‐RBD‐NG compared to S‐RBD alone (Figure 3C). DCs and macrophages in the inguinal lymph nodes were further analyzed by flow cytometry, and enhanced uptake of S‐RBD‐NG was observed in these cells (Figure 3D and Figure S9, Supporting Information). The mechanism underlying the enhanced uptake of S‐RBD‐NG by lymph nodes and APCs remains unclear. Since the immune system has evolved the ability to capture and process nanosized virus‐like particles,[ ] the nanoparticles could be filtered and accumulate in the lymphoid organs (e.g., liver, spleen, and lymph nodes), followed by rapid uptake and phagocytosis by APCs, the major cell types responsible for capturing these nanoparticles in a size‐dependent manner.[ ] In addition, the change in surface charge may play an important role in lymph node uptake.[ ]

Figure 3

The lymph node targeting ability of S‐RBD and S‐RBD‐NG. A) Schematic representation of the characterization process. B) Ex vivo imaging analysis of the inguinal lymph nodes. C) Quantitative analysis of the accumulation of S‐RBD and S‐RBD‐NG in inguinal lymph nodes. D) Quantitative analysis of the cellular uptake of S‐RBD or S‐RBD‐NG by DCs and macrophages in vivo. Data are presented as mean ± SEM. n = 3.

Encouraged by the lymph node targeting and DCs/macrophage uptake results, we next examined the immunogenicity of S‐RBD‐NG in vivo. C57BL/6N mice were immunized intramuscularly with PBS, S‐RBD (50 µg per mouse), or S‐RBD‐NG (50 µg per mouse) in the presence or the absence of an aluminum hydroxide adjuvant (100 µg per mouse), one of the most commonly used adjuvants for vaccine development. Mice were further boosted with the same dosage on days 14 and 28, and sera were collected one week after each immunization (Figure  ). S‐RBD‐specific serum IgG was detected using ELISA, and the titers were calculated. One week after the first immunization, the IgG titers were still below our detection limit (lower than the lowest dilution factor 50, data not shown) for all groups. After the second round of immunization, S‐RBD‐specific serum IgG titers were increased to ≈104 for S‐RBD‐NG treated groups, both in the presence and absence of aluminum hydroxide adjuvant (Figure 4B,C). Notably, after the third round of immunization, the titers for the S‐RBD‐NG‐treated group reached ≈105, while the S‐RBD monomer‐treated group had a titer less than 104. Quantitative comparison showed that S‐RBD‐NG induced 27.6‐ and 8.3‐fold higher titers than the S‐RBD monomer in the absence or presence of aluminum hydroxide adjuvant. Taken together, these results showed that S‐RBD‐NG possessed higher immunogenicity than S‐RBD, and disassembly of this internalized pro‐antigen elicited more potent and rapid immune responses.

Figure 4

Immunization and detection of antibody titers in mice. A) Schematic representation of the immunization procedure. B) Detection of S‐RBD‐specific IgG antibodies from mouse sera collected on day 21 using ELISA. C) Titer of S‐RBD‐specific IgG antibodies calculated from (B). D) Detection of S‐RBD‐specific IgG antibodies from mouse sera collected on day 35 using ELISA. E) Titer of S‐RBD‐specific IgG antibodies calculated from (D). F) Detection of S‐RBD‐specific IgG antibodies in sera from mice immunized with S‐RBD‐NG and Pam3CSK4 on day 21 and 35 using ELISA. G) Titer of S‐RBD‐specific IgG antibodies calculated from (F). Data are presented as mean ± SEM. n = 6.

Next, we examined whether the toll‐like receptor 1/2 agonist Pam3CSK4, another frequently used adjuvant in vaccine development, could further boost the immunogenicity of S‐RBD‐NG.[ ] Indeed, coadministration of S‐RBD‐NG with Pam3CSK4 stimulated similar but more potent immune responses (Figure 4F,G). The S‐RBD‐specific IgG titer reached ≈106 after the third round of immunization. This suggested that the vaccination titer of S‐RBD NGs could be further improved by optimizing the adjuvant used.

Since blocking the interaction between spike protein and ACE2 is crucial for preventing SARS‐CoV‐2's entry into host cells, we investigated whether the sera from the immunized mice were able to inhibit this interaction. A competitive ELISA strategy was employed in which the sera were used to compete with hACE2 for binding to the immobilized S‐RBD. Indeed, the sera from S‐RBD‐NG‐immunized groups (either in the absence or presence of adjuvant) efficiently blocked the S‐RBD‐hACE2 interaction (Figure S10, Supporting Information), which was consistent with the aforementioned titer measurement. Therefore, S‐RBD‐NG induced the development of specific antibodies that could target and block the interaction between S‐RBD and hACE2.

Finally, we used the pseudovirus to further test the utility of S‐RBD‐NG as a pro‐antigen for subunit vaccine development for SARS‐CoV‐2 neutralization. The SARS‐CoV‐2 pseudovirus contains the spike protein shell and harbors a luciferase gene as a reporter (termed Spike‐PV‐Luc). The COS7 cell line stably expressing hACE2 (COS7‐hACE2) was generated to mimic human cells. Expression of hACE2 was first validated by immunofluorescence (Figure S11, Supporting Information). The neutralization activity of the sera from different immunized mice was assessed by monitoring the transduction efficiency of Spike‐PV‐Luc. Indeed, the inhibition of Spike‐PV‐Luc transduction by immunized sera was consistent with the titer measured (Figure  ). In particular, sera from mice immunized with S‐RBD‐NG and Pam3CSK4 almost completely inhibited pseudovirus entry under both dilution factors. Sera from S‐RBD‐NG immunized mice inhibited pseudovirus transduction in a dilution‐dependent manner, both in the presence or absence of the aluminum hydroxide adjuvant. In contrast, no inhibition was observed using sera from PBS‐ or S‐RBD immunized mice. Interestingly, when the sera were used at a higher concentration (20‐fold dilution), the pseudovirus transduction was enhanced. This may be due to the antibody‐dependent enhancement,[ ] which indicated that antibodies from SARS‐CoV‐2 infected blood may facilitate virus entry. To further confirm the results, another spike pseudovirus harboring the gfp gene as the reporter (termed Spike‐PV‐GFP) was prepared. Inhibition of spike‐PV‐GFP transduction into COS7‐hACE2 cells by immunized sera was observed by confocal microscopy imaging (Figure 5B). The sera from mice immunized with S‐RBD‐NG were found to neutralize the SARS‐CoV‐2 pseudovirus.

Figure 5

Neutralization of SARS‐CoV‐2 spike pseudovirus using immunized mouse sera. A) Transduction inhibition of the Spike‐PV‐Luc by different sera. Spike‐PV‐Luc was pre‐incubated with sera from different groups at 1:40 or 1:20 dilution and then added to COS7‐hACE2. Transduction efficiency was assessed by luciferase reporter. Data are presented as mean ± SEM. n = 3 or 4. B) Transduction inhibition of Spike‐PV‐GFP by different sera. Spike‐PV‐GFP was pre‐incubated with sera from different groups at 1:20 dilution and then added to COS7‐hACE2. Transduction efficiency was assessed by confocal microscopy imaging. Scale bar: 50 µm.

To show that our pro‐antigen strategy can be generally applicable to other viruses, we formulated the recombinant S1 subunit of SARS‐CoV‐1 as NGs and the uptake by RAW 264.7 cells was validated. Indeed, the intracellular uptake of the resulting SARS‐CoV‐S1‐NG was greatly enhanced (Figure S12, Supporting Information). Since many other viruses such as Ebola virus also depend on the envelope‐attached glycoproteins for entering host cells, we envision that our pro‐antigen strategy may be extended to these viruses for subunit vaccine development.

Conclusion

In conclusion, we developed a generally applicable pro‐antigen strategy by employing the reversibly formulated S‐RBD‐NG to enhance the immunogenicity of SARS‐CoV‐2 spike proteins. S‐RBD‐NG showed improved lymph node targeting and accumulation in APCs, which can be rapidly converted into S‐RBD monomers after internalization, leading to more potent immune responses during in vivo immunization. These results demonstrated the advantages of S‐RBD‐NG over S‐RBD monomer for future subunit vaccine development. Notably, S‐RBD‐NG alone was able to induce rapid and potent immune responses, which offers the possibility of developing subunit vaccine without the use of adjuvants. The immunized sera were further shown to block the interaction between the spike protein and hACE2, which is crucial for virus entry into host cells. Finally, in the pseudovirus neutralization assay, sera from S‐RBD‐NG‐immunized groups effectively neutralized the pseudovirus in a concentration‐dependent manner. The S‐RBD‐NG‐based pro‐antigen strategy within the lymph node niche can elicit more rapid and potent immune responses and may serve as a potential subunit vaccine candidate against SARS‐CoV‐2.

Conflict of Interest

L. Chen, J. Lin, and P. R. Chen are inventors of the patent related to this manuscript.

Supporting Information

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