Enhancement of Immune Response of Bioconjugate Nanovaccine by Loading of CpG through Click Chemistry

CpG is a widely used adjuvant that enhances the cellular immune response by entering antigen-presenting cells and binding with receptors. The traditional physical mixing of the antigen and CpG adjuvant results in a low adjuvant utilization rate. Considering the efficient delivery capacity of nanovaccines, we developed an attractive strategy to covalently load CpG onto the nanovaccine, which realized the co-delivery of both CpG and the antigen. Briefly, the azide-modified CpG was conjugated to a bioconjugate nanovaccine (NP-OPS) against Shigella flexneri through a simple two-step reaction. After characterization of the novel vaccine (NP-OPS-CpG), a series of in vitro and in vivo experiments were performed, including in vivo imaging, lymph node sectioning, and dendritic cell stimulation, and the results showed that more CpG reached the lymph nodes after covalent coupling. Subsequent flow cytometry analysis of lymph nodes from immunized mice showed that the cellular immune response was greatly promoted by the nanovaccine coupled with CpG. Moreover, by analyzing the antibody subtypes of immunized mice, NP-OPS-CpG was found to further promote a Th1-biased immune response. Thus, we developed an attractive method to load CpG on a nanovaccine that is simple, convenient, and is especially suitable for immune enhancement of vaccines against intracellular bacteria.


Introduction
Vaccines are of great significance to human health, and currently, vaccination has become increasingly important for disease management given its wide applicability and long-term protection capability [1,2]. From the early inactivated or attenuated live vaccines to the current subunit vaccines, as well as the mRNA vaccines against COVID-19, many safer and more efficient vaccine systems are being continuously developed [3]. Subunit vaccines have weak immunogenicity that makes it difficult for them to stimulate an effective antibody response [4] and will need further modifications to overcome this bottleneck. In recent decades, many advanced delivery systems exhibiting both high efficacy and safety were developed. Antigens or epitopes can now be loaded efficiently in many ways to realize the targeted delivery. Nanovaccines, which have a pronounced ability to affect lymph node drainage and immune activation [2], have received widespread attention. In recent years, various nanoparticles, such as inorganic nanoparticles (NPs), inorganic and organic hybrid NPs, organic NPs, and proteinaceous NPs, have been used to develop bacterial vaccines and have shown powerful effects [2,5]. Prophylactic vaccines using proteinaceous nanomaterials (e.g., virus-like particles and ferritin) with a higher safety and biocompatibility are being widely studied [6]. With the development of synthetic biology, modular self-assembled nanoparticles are also being explored in vaccine design. In our previous study, we successfully prepared self-assembled nanocarriers by the fusion

Bacterial Strains and Growth Conditions
NP-OPS was expressed in Shigella 301DWP containing the pET28a-pglL-CTBtri4573 plasmid as previously described [7]. Bacteria were cultured in Luria-Bertani (LB) medium at 37 • C (220 rpm). For expression, cells were cultured to an optical density value of 0.6-0.8 at 600 nm and induced with a final concentration of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) to express NP-OPS. The culture then continued to incubate at 30 • C for 14 h (220 rpm).

Preparation of NP-OPS-CpG via Click Chemistry
Based on the principle of the covalent binding of free amino groups in proteins to succinimide, we mixed NP-OPS with succinimide-PEG 4 -alkyne at 25 • C for 2 h. Then, the mixture was dialyzed in phosphate-buffered saline (PBS) at 4 • C for 2 days. The PBS was replaced every 6 h with a final change using ddH 2 O to obtain pure NP-OPS-alkyne. CpG and its modified products were purchased from Generay. Based on the principle of the Cu-catalyzed azide-alkyne cycloaddition reaction, NP-OPS-CpG was obtained by combining lyophilized NP-OPS-alkyne with azide-modified CpG2006 (N 3 -CpG, CpG2006: 5 -TCGTCGTTTTTGTCGTTGTCGTT-3 ) by using a Click-iT protein buffer kit (Thermo Fisher, Waltham, MA, USA). Finally, the purified NP-OPS-CpG was obtained using sizeexclusion chromatography through a Superdex-200 column (GE Healthcare, Chicago, IL, USA) in a mobile phase consisting of PBS, as described previously.

Coomassie Blue Staining and Western Blotting
The preparation of protein samples was carried out as described previously with slight modifications [7]. The purified NP-OPS, NP-OPS-alkyne, and NP-OPS-CpG were verified with the Coomassie blue staining method. The glycoprotein samples were detected through Western blotting with anti-6 × His antibody (Abmart, Shanghai, China) and anti-S. flexneri OPS-specific serum (Denka Seiken, Tokyo, Japan).

Stimulation of DC2.4 Mouse Dendritic Cell Line (DC2.4s) by Vaccines In Vitro
The nucleic acid concentration of the sample NP-OPS-CpG was measured by NanoDrop TM 2000 (Thermo Fisher, Waltham, MA, USA). CpG was added to the NP-OPS (without alkyne) to obtain the sample of NP-OPS+CpG. To ensure the same amount of nucleic acid in treat, the CpG concentration in NP-OPS+CpG sample was measured to be consistent with that of NP-OPS-CpG. Untreated DC2.4s were cultured to 100,000 cells/well at 37 • C with 5% CO 2 . DC2.4s were stimulated with PBS, CpG Cy5 , NP-OPS mixed with CpG Cy5 (NP-OPS+CpG Cy5 ), and NP-OPS-coupled CpG Cy5 (NP-OPS-CpG Cy5 ). Each treat contained 50 ng CpG Cy5 . After incubating for 6 h, 12 h, or 24 h, DC2.4s were digested with trypsin, and free cells were obtained by centrifugation at 4 • C (500× g, 5 min). Cells were centrifuged and resuspended in 100 µL of cold staining buffer (eBioscience, San Diego, CA, USA), and this step was repeated twice. The washed cells were filtered through a 200-mesh screen to obtain samples for flow cytometry. Finally, Cy5-labeled DC2.4s were analyzed using a CytoFLEX LX flow cytometer (Beckman, Brea, CA, USA).

Lymph Node Imaging Assay
Mice were randomly divided into three groups (CpG Cy7 , NP-OPS+CpG Cy7 , and NP-OPS-CpG Cy7 ). Mice were injected in the tail base with samples that had a consistent fluorescence intensity. Mouse lymph node fluorescence signals at different time points (0 h, 6 h, 12 h, and 24 h after injection) were measured by an IVIS spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA).

Mouse Immunization
Isonucleic acid concentrations of NP-OPS-CPG and NP-OPS+CpG were prepared as described above. Specific-pathogen-free female BALB/c mice (6-8 weeks old) were purchased from Spaifer and were randomly divided into 4 groups (10 mice in each group).
Mice were subcutaneously injected with 100 µL of PBS, NP-OPS, NP-OPS+CpG, or NP-OPS-CpG with a polysaccharide dose of 2.5 µg/mouse on days 0, 14, and 28. Corresponding CpG in both NP-OPS+CpG and NP-OPS-CpG groups was 120 ng/mouse. One week after the last immunization, blood samples were taken by tail clipping, and serum was stored at 4 • C. All animal experiments were approved and conducted by the institutional guidelines of the Academy of Military Medical Sciences and with the approval of the Institutional Animal Care and Use Committee (Approval Code IACUC-DWZX-2021-073).

T Cell Immune Response Induced by the Vaccines In Vivo
Mice were grouped and immunized as described previously with slight modifications [7]. BALB/c mice were immunized with one of three formulations (NP-OPS, NP-OPS+CpG, and NP-OPS-CpG), and draining lymph nodes (dLNs) of each mouse were individually collected three days post-vaccination and triturated into single cell suspension. Then, the cells were stained with APC-conjugated anti-mouse CD3. After fixation and permeabilization, the cells were further stained with FITC-conjugated anti-mouse Ki 67 antibody and analyzed by flow cytometry.
Five days after the third immunization, the mice were sacrificed to obtain lymph nodes. Flow cytometry samples were prepared as described above. Cells were stained with the following antibodies: APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, and PE-conjugated anti-mouse CD8. CD3 + , CD4 + , and CD8 + cells were analyzed by a CytoFLEX LX flow cytometer (Beckman, Brea, CA, USA). All flow cytometry antibodies were purchased from eBioscience, San Diego, CA, USA.

ELISA
S. flexneri 2a lipopolysaccharide (LPS) (100 µg/well) was diluted with a coating solution and plated onto 96-well plates that were incubated overnight at 4 • C. The plates were washed 3 times with PBST (PBS with 0.05% Tween) and blocked with 5% skim milk at 37 • C for 2 h. After washing the plates 3 times, serum diluted with a holding solution in a 2-fold serial ratio was added to the plates and incubated at 37 • C for 1 h. After again washing 3 times, 100 µL of HRP-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) (1:15,000) was added and incubated at 37 • C for 1 h. After washing the plate again, 100 µL of tetramethylbenzidine solution (CWBio, Beijing, China) was added for the color reaction. Finally, the absorbance value of each well at 450 nm was measured after adding the termination solution.

Immune Effects of the NP-OPS-CpG in an S. flexneri 2a Infection Model
Next, 14 days after 3 immunizations, mice were challenged with 2.5 × 10 7 CFU S. flexneri 2a by intraperitoneal injection (i.p.), and the survival of each group of mice was monitored continuously for 14 days (n = 10).

Statistical Analysis
All data in these experiments were analyzed by GraphPad Prism 7.0 statistical software (GraphPad Inc, San Diego, CA, USA). The data were analyzed using one-way ANOVA and t-test. Results were expressed as means ± SDs. Values of p < 0.05 were considered statistically significant (**** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05).

Conjugation of NP-OPS and CpG
The NP-OPS-CpG conjugate vaccines were prepared as described in the experimental protocol ( Figure 1a). First, to modify the alkyne group on NP-OPS, NP-OPS was incubated with succinimide-PEG 4 -alkyne at 25 • C for 2 h. After removing unbound succinimide-PEG 4 -alkyne through dialysis, NP-OPS containing the alkyne group was further incubated with N 3 -CpG at 25 • C for 20 min. Then, NP-OPS-CpG was obtained by size-exclusion chromatography. After separating samples from each step in the process using SDS-PAGE, results from the Coomassie blue staining and Western blot with an anti-6 × His tag antibody and anti-S. flexneri OPS serum showed that the typical NP-OPS ladder had an obvious upward migration after each step, indicating the successful loading of succinimide-PEG 4alkyne and CpG separately ( Figure 1b). As expected, the size-exclusion chromatography results showed that the retention volume of NP-OPS-CpG was 8-11 mL, about 30% of the column volume, indicating that NP-OPS-CpG was still in the form of a polymer (Figure 1c). To further determine the combination, different concentrations of FAM-labeled N 3 -CpG were incubated with a certain amount of NP-OPS-alkyne, as described above. After separating by SDS-PAGE, samples were analyzed by Coomassie blue staining and observed under ultraviolet light. The results showed that with an increased concentration of CpG, the fluorescence intensity of the NP-OPS band increased, suggesting that CpG coupled with NP-OPS ( Figure 1d).

Conjugation of NP-OPS and CpG
The NP-OPS-CpG conjugate vaccines were prepared as described in the experimental protocol (Figure 1a). First, to modify the alkyne group on NP-OPS, NP-OPS was incubated with succinimide-PEG4-alkyne at 25 °C for 2 h. After removing unbound succinimide-PEG4-alkyne through dialysis, NP-OPS containing the alkyne group was further incubated with N3-CpG at 25 °C for 20 min. Then, NP-OPS-CpG was obtained by sizeexclusion chromatography. After separating samples from each step in the process using SDS-PAGE, results from the Coomassie blue staining and Western blot with an anti-6 × His tag antibody and anti-S. flexneri OPS serum showed that the typical NP-OPS ladder had an obvious upward migration after each step, indicating the successful loading of succinimide-PEG4-alkyne and CpG separately ( Figure 1b). As expected, the size-exclusion chromatography results showed that the retention volume of NP-OPS-CpG was 8-11 mL, about 30% of the column volume, indicating that NP-OPS-CpG was still in the form of a polymer (Figure 1c). To further determine the combination, different concentrations of FAM-labeled N3-CpG were incubated with a certain amount of NP-OPS-alkyne, as described above. After separating by SDS-PAGE, samples were analyzed by Coomassie blue staining and observed under ultraviolet light. The results showed that with an increased concentration of CpG, the fluorescence intensity of the NP-OPS band increased, suggesting that CpG coupled with NP-OPS ( Figure 1d).

Analysis of the NP-OPS-CpG Bioconjugate Nanovaccine
Previous results showed that NP-OPS existed in the form of nanoparticles [7]. To determine whether the coupling of CpG affected the formation of particles, purified NP-OPS-CpG was analyzed using transmission electron microscopy (TEM). The results showed that NP-OPS-CpG was about 20-30 nm in diameter ( Figure 2a). Dynamic light scattering (DLS) results also revealed that NP-OPS-CpG was monodisperse and was about 29.76 nm, which was in line with the TEM result and consistent with that before coupling

Analysis of the NP-OPS-CpG Bioconjugate Nanovaccine
Previous results showed that NP-OPS existed in the form of nanoparticles [7]. To determine whether the coupling of CpG affected the formation of particles, purified NP-OPS-CpG was analyzed using transmission electron microscopy (TEM). The results showed that NP-OPS-CpG was about 20-30 nm in diameter (Figure 2a). Dynamic light scattering (DLS) results also revealed that NP-OPS-CpG was monodisperse and was about 29.76 nm, which was in line with the TEM result and consistent with that before coupling due to the small molecular weight of CpG. The polydispersity index (PDI) was about 0.2698 (slightly higher than 0.2), which may be attributed to the nonuniform length of polysaccharide antigens on the nanoparticle. Although the NP-OPS was almost uncharged due to the coverage of polysaccharides which had no charge itself, the coupling of CpG made it carry negative charges, also suggesting that CpG was successfully coupled to the surface of NP-OPS ( Figure S1). In addition, by measuring the concentrations of nucleic acid and proteins in NP-OPS-CpG, respectively, we calculated that there were about 40 CpG on each particle. Then, we analyzed the stability of NP-OPS-CpG and found that it maintained stability after being stored at 37 • C for at least 7 days (Figure 2c). charged due to the coverage of polysaccharides which had no charge itself, the coupling of CpG made it carry negative charges, also suggesting that CpG was successfully coupled to the surface of NP-OPS ( Figure S1). In addition, by measuring the concentrations of nucleic acid and proteins in NP-OPS-CpG, respectively, we calculated that there were about 40 CpG on each particle. Then, we analyzed the stability of NP-OPS-CpG and found that it maintained stability after being stored at 37 °C for at least 7 days (Figure 2c).

Evaluation of CpG Lymph Node Targeting
One of the advantages of nanovaccines is efficient lymph node drainage, enabling more antigens to reach immune organs. Because the CpG in our design was covalently coupled to the bioconjugate nanovaccine, it would also be delivered, in theory. To confirm this, Cy5-labeled CpG was coupled with NP-OPS, and the three treatments (CpGCy5, NP-OPS+CpGCy5, or NP-OPS-CpGCy5) were injected into the tail base of BALB/c mice. DLNs were taken from each mouse 6 h post-injection, and the fluorescence imaging results of dLNs sections showed that stronger CpG signals were detected in NP-OPS-CpG-immunized mice compared to the other mice ( Figure 3a). Then, Cy7-labeled CpG was coupled to NP-OPS, and mice were injected as above. In vivo imaging results showed that the intensity of the signal for CpG alone at the site of the dLNs was barely detected at any time point. The signal for NP-OPS+CpGCy7 revealed an obvious accumulation pattern in the dLNs, possibly due to a small amount of non-specific binding. In contrast, dramatic increases in lymph node accumulation were observed for NP-OPS-CpGCy7, which was attributed to the co-delivery with the nanovaccine (Figure 3b). Furthermore, the analysis of the total signal intensity throughout the time course experiment revealed an 11 times increase in the lymph-node-specific accumulation of NP-OPS-CpG over CpG (Figure 3c).

Evaluation of CpG Lymph Node Targeting
One of the advantages of nanovaccines is efficient lymph node drainage, enabling more antigens to reach immune organs. Because the CpG in our design was covalently coupled to the bioconjugate nanovaccine, it would also be delivered, in theory. To confirm this, Cy5-labeled CpG was coupled with NP-OPS, and the three treatments (CpG Cy5 , NP-OPS+CpG Cy5 , or NP-OPS-CpG Cy5 ) were injected into the tail base of BALB/c mice. DLNs were taken from each mouse 6 h post-injection, and the fluorescence imaging results of dLNs sections showed that stronger CpG signals were detected in NP-OPS-CpG-immunized mice compared to the other mice ( Figure 3a). Then, Cy7-labeled CpG was coupled to NP-OPS, and mice were injected as above. In vivo imaging results showed that the intensity of the signal for CpG alone at the site of the dLNs was barely detected at any time point. The signal for NP-OPS+CpG Cy7 revealed an obvious accumulation pattern in the dLNs, possibly due to a small amount of non-specific binding. In contrast, dramatic increases in lymph node accumulation were observed for NP-OPS-CpG Cy7 , which was attributed to the co-delivery with the nanovaccine (Figure 3b). Furthermore, the analysis of the total signal intensity throughout the time course experiment revealed an 11 times increase in the lymph-node-specific accumulation of NP-OPS-CpG over CpG (Figure 3c).

Analysis of CpG Phagocytosis by DC2.4s
Having confirmed the efficient lymph node drainage of CpG loaded onto the nanovaccine, we further analyzed the endocytic activity of DCs in different forms of CpG. DC2.4 is a mouse bone marrow dendritic cell line established with C57BL/6 mouse bone marrow cells transfected by the V-myc and V-raf genes and used to simulate the function

Analysis of CpG Phagocytosis by DC2.4s
Having confirmed the efficient lymph node drainage of CpG loaded onto the nanovaccine, we further analyzed the endocytic activity of DCs in different forms of CpG. DC2.4 is a mouse bone marrow dendritic cell line established with C57BL/6 mouse bone marrow cells transfected by the V-myc and V-raf genes and used to simulate the function of APC in several studies [30,38]. By stimulating DC2.4s with CpG Cy5 , NP-OPS+CpG Cy5 , or NP-OPS-CpG Cy5 , the amount of CpG phagocytosed by DC2.4s was analyzed by flow cytometry. The results showed that although Cy5 + in the DC2.4s in the CpG Cy5 and NP-OPS+CpG Cy5 treatment groups increased at the three time points, the greatest Cy5 signals were detected in the NP-OPS-CpG Cy5 group (Figure 4a,b). Particularly when incubated for 24 h, the phagocytic efficiency was about four times higher than that of CpG alone ( Figure S2).  . Data are presented as means ± SD. Each group was compared with NP-OPS-CpGCy5 using one-way ANOVA: **** p < 0.0001, *** p < 0.001, and ** p < 0.01.

T Cell Proliferation and Differentiation Induced by the Nanovaccines
To explore the effects of the different vaccines on the proliferation and differentiation of T cells, BALB/c mice were immunized with NP-OPS, NP-OPS+CpG, or NP-OPS-CpG. Three days after immunization, mice were sacrificed, the dLNs were removed, and Ki67 + and CD3 + cells in dLNs were analyzed by flow cytometry. The results showed the percentage of Ki67-positive T cells was significantly increased in the NP-OPS-CpG-treated group compared with that of other groups (Figure 5a). In addition, after three immunizations at two-week intervals, mice were sacrificed five days after the last injections, and dLNs were removed. Flow cytometry results showed that NP-OPS-CpG-injected mice had a significant increase in the ratio of CD3 + cells ( Figure S3). The proportion of CD4 + T cells and CD8 + T cells in lymph nodes was also analyzed, and the results showed that the NP-OPS-CpG-injected mice had the greatest increase in both subtypes (Figure 5b,c). In particular, CD8 + levels in the NP-OPS-CpG group increased more than in the NP-OPS+CpG group (Figure 5c). . Data are presented as means ± SD. Each group was compared with NP-OPS-CpG Cy5 using one-way ANOVA: **** p < 0.0001, *** p < 0.001, and ** p < 0.01.

T Cell Proliferation and Differentiation Induced by the Nanovaccines
To explore the effects of the different vaccines on the proliferation and differentiation of T cells, BALB/c mice were immunized with NP-OPS, NP-OPS+CpG, or NP-OPS-CpG. Three days after immunization, mice were sacrificed, the dLNs were removed, and Ki67 + and CD3 + cells in dLNs were analyzed by flow cytometry. The results showed the percentage of Ki67positive T cells was significantly increased in the NP-OPS-CpG-treated group compared with that of other groups (Figure 5a). In addition, after three immunizations at two-week intervals, mice were sacrificed five days after the last injections, and dLNs were removed. Flow cytometry results showed that NP-OPS-CpG-injected mice had a significant increase in the ratio of CD3 + cells ( Figure S3). The proportion of CD4 + T cells and CD8 + T cells in lymph nodes was also analyzed, and the results showed that the NP-OPS-CpG-injected mice had the greatest increase in both subtypes (Figure 5b,c). In particular, CD8 + levels in the NP-OPS-CpG group increased more than in the NP-OPS+CpG group (Figure 5c). . Lymph nodes were obtained from mice three days after immunization, and CD3 + and CD3 + Ki67 + cells were analyzed by flow cytometry. (b,c) NP-OPS, NP-OPS+CpG, and NP-OPS-CpG were injected into the tail base of mice separately (n = 3). Lymph nodes were obtained from mice five days after the third immunization, and CD4 + (b) and CD8 + (c) cells were analyzed by flow cytometry (n = 3). Each group was compared with NP-OPS-CpG using one-way ANOVA: *** p < 0.001; ** p < 0.001, and * p < 0.05.

Antibody Response and Protective Effect in NP-OPS-CpG Immunized Mice
To evaluate the antibody response induced by NP-OPS-CpG, BALB/c mice were immunized with 1 of 4 treatments (PBS control, NP-OPS, NP-OPS+CpG, or NP-OPS-CpG) on days 0, 14, and 28. Blood was sampled on day 38 to facilitate the quantitation of antibodies against S. flexneri 2a LPS. The bacterial pathogen challenge was administered on day 42, followed by the monitoring of mouse survival (Figure 6a). ELISA results revealed that NP-OPS-CpG induced a higher IgG titer, although it had no statistical significance (Figure 6b). Subsequently, titers of the IgG subtype (IgG1 and IgG2a) were measured, and the results showed that the NP-OPS-CpG-treated group induced significantly higher IgG2a titers, suggesting further promotion of a Th1-biased immune response (Figure 6c). In addition, by calculating the ratio of IgG1 and IgG2a of each group, it was found that NP-OPS coupled with CpG revealed a significantly lower ratio than that of the NP-OPS+CpG group (Figure 6c). These results indicated that although physical mixing of CpG improved the Th1 immune response, coupling was more conducive to establishing a balance favoring a Th1-biased immune response. Then, mice were injected intraperitoneally with a dose of 2.5 × 10 7 CFU per mouse of S. flexneri 2a strain 14 days after the third immunization, and the survival of each group of mice was observed. All the mice in the . Lymph nodes were obtained from mice three days after immunization, and CD3 + and CD3 + Ki67 + cells were analyzed by flow cytometry. (b,c) NP-OPS, NP-OPS+CpG, and NP-OPS-CpG were injected into the tail base of mice separately (n = 3). Lymph nodes were obtained from mice five days after the third immunization, and CD4 + (b) and CD8 + (c) cells were analyzed by flow cytometry (n = 3). Each group was compared with NP-OPS-CpG using one-way ANOVA: *** p < 0.001; ** p < 0.001, and * p < 0.05.

Antibody Response and Protective Effect in NP-OPS-CpG Immunized Mice
To evaluate the antibody response induced by NP-OPS-CpG, BALB/c mice were immunized with 1 of 4 treatments (PBS control, NP-OPS, NP-OPS+CpG, or NP-OPS-CpG) on days 0, 14, and 28. Blood was sampled on day 38 to facilitate the quantitation of antibodies against S. flexneri 2a LPS. The bacterial pathogen challenge was administered on day 42, followed by the monitoring of mouse survival (Figure 6a). ELISA results revealed that NP-OPS-CpG induced a higher IgG titer, although it had no statistical significance (Figure 6b). Subsequently, titers of the IgG subtype (IgG1 and IgG2a) were measured, and the results showed that the NP-OPS-CpG-treated group induced significantly higher IgG2a titers, suggesting further promotion of a Th1-biased immune response (Figure 6c). In addition, by calculating the ratio of IgG1 and IgG2a of each group, it was found that NP-OPS coupled with CpG revealed a significantly lower ratio than that of the NP-OPS+CpG group (Figure 6c). These results indicated that although physical mixing of CpG improved the Th1 immune response, coupling was more conducive to establishing a balance favoring a Th1-biased immune response. Then, mice were injected intraperitoneally with a dose of 2.5 × 10 7 CFU per mouse of S. flexneri 2a strain 14 days after the third immunization, and the survival of each group of mice was observed. All the mice in the PBS group died rapidly in the first two days, and all mice in the other three groups were alive (Figure 6d). The results indicated that by coupling with CpG, NP-OPS maintained efficient prophylactic effects against infection.
PBS group died rapidly in the first two days, and all mice in the other three groups were alive (Figure 6d). The results indicated that by coupling with CpG, NP-OPS maintained efficient prophylactic effects against infection.

Discussion
In this study, we developed an attractive strategy for producing a bioconjugate nanovaccine loaded with a CpG adjuvant. Different from traditional physical mixing, the covalent coupling of CpG and NP-OPS realized the co-delivery of an antigen and CpG through nano-carriers. CpG was rapidly drained to the lymph nodes by using nano-carriers and was easily engulfed by antigen-presenting cells. Subsequently, the cellular immune response was greatly enhanced. Therefore, we provided here a novel method for loading CpG onto a nanovaccine. This method is simple, convenient, and is especially suitable for the immune enhancement of intracellular bacterial vaccines.
In recent years, the CpG adjuvant has been widely used in vaccine research. Particularly for COVID-19 vaccines, CpG is often used together with a classical aluminum adjuvant [39]. By adsorbing to an aluminum salt through electrostatic action, CpG utilized the storage effect of the aluminum adjuvant and was beneficial for activating a cellular immune response. However, this compatibility may not be suitable for nanovaccines. Generally, one of the advantages of nanovaccines is the size-related ability to rapidly drain to and accumulate in lymph nodes [40]. As previously reported, 15-100 nm is the optimal size of vaccines for direct homing to draining lymph nodes [41]. Thus, this advantage is weakened if an aluminum adjuvant is added. Furthermore, our previous study also showed that the addition of an aluminum adjuvant to the bioconjugate nanovaccine did not further improve the antibody response [8]. Therefore, coupling with CpG is appropriate for improving the bioconjugate nanovaccine response. This conjugation not only maintained the size advantage of the vaccine, but also realized the co-delivery of CpG and the antigen. In our results, more CpG, when coupled with NP-OPS, reached the lymph nodes with a better uptake by DCs compared to a physical mixture with CpG; thus, the coupling strategy was more efficient and conducive to stimulating a cellular immune response. Each group was compared with NP-OPS-CpG using one-way ANOVA: **** p < 0.0001; ** p < 0.01; and * p < 0.05.

Discussion
In this study, we developed an attractive strategy for producing a bioconjugate nanovaccine loaded with a CpG adjuvant. Different from traditional physical mixing, the covalent coupling of CpG and NP-OPS realized the co-delivery of an antigen and CpG through nano-carriers. CpG was rapidly drained to the lymph nodes by using nano-carriers and was easily engulfed by antigen-presenting cells. Subsequently, the cellular immune response was greatly enhanced. Therefore, we provided here a novel method for loading CpG onto a nanovaccine. This method is simple, convenient, and is especially suitable for the immune enhancement of intracellular bacterial vaccines.
In recent years, the CpG adjuvant has been widely used in vaccine research. Particularly for COVID-19 vaccines, CpG is often used together with a classical aluminum adjuvant [39]. By adsorbing to an aluminum salt through electrostatic action, CpG utilized the storage effect of the aluminum adjuvant and was beneficial for activating a cellular immune response. However, this compatibility may not be suitable for nanovaccines. Generally, one of the advantages of nanovaccines is the size-related ability to rapidly drain to and accumulate in lymph nodes [40]. As previously reported, 15-100 nm is the optimal size of vaccines for direct homing to draining lymph nodes [41]. Thus, this advantage is weakened if an aluminum adjuvant is added. Furthermore, our previous study also showed that the addition of an aluminum adjuvant to the bioconjugate nanovaccine did not further improve the antibody response [8]. Therefore, coupling with CpG is appropriate for improving the bioconjugate nanovaccine response. This conjugation not only maintained the size advantage of the vaccine, but also realized the co-delivery of CpG and the antigen. In our results, more CpG, when coupled with NP-OPS, reached the lymph nodes with a better uptake by DCs compared to a physical mixture with CpG; thus, the coupling strategy was more efficient and conducive to stimulating a cellular immune response.
In our study, CpG was covalently coupled with the nanovaccine through a succinimide-PEG 4 -alkyne-bridge. Many other coupling modes have been developed. At present, the most widely used coupling agent is SMCC, which contains N-hydroxysuccinimide active ester and maleimide. The two active groups of SMCC couple CpG with a protein antigen. However, because disulfide bonds need to be opened during the crosslinking process, SMCC is not suitable for antigens containing more than two cysteines [35]. Our study selected succinimide-PEG 4 -alkyne as a connector for which active groups can complete the reaction quickly under mild conditions [42]. At the same time, no influence on the protein structure was found. Therefore, it was suitable for proteinaceous nano-carriers. In addition, the protein HUH also binds to CpG. By fusion expression with protein antigens, HUH can serve as a bridge to load CpG. Considering the influence of HUH expression on nanostructures, this strategy is more suitable for monomer protein carriers.
Our results have shown that the covalent coupling of CpG and the delivery vector was more efficient than the physical mixing to induce a cellular immune response. However, there still were some possible limitations in this study. For example, to better evaluate the effect of the NP-OPS-CpG conjugate vaccine, a group treated with a traditional adjuvant (such as aluminum) could be added as a control to determine the relative efficacy. In addition, in our research, we focused on the vaccine's efficacy against the S. flexneri infection. Considering that the active groups we selected can complete the reaction quickly under mild conditions, universal applicability could be explored, and the immune enhancement effect of serious vaccines against other pathogens (especially intracellular bacteria) can also be further measured. Moreover, increasing the sample size in future studies would strengthen the findings. In addition, because this coupling method increases the CpG utilization rate, the dosage of CpG can be reduced and compared with the current dosage, thus reducing the production cost and the side effects. This method also provides direction for the research of a new generation of self-adjuvant vaccines. Moreover, through modifications of nanostructures and amino acids in the future, more binding sites can be designed to realize controllable CpG loading.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.