Preparation and characterization of CMR-PLGA/OVA NVs
To targeting delivery of antigens, PLGA/OVA NVs were fabricated via the double emulsion process, and the CpG-Man-RBC membrane was further coated on the surfaces of PLGA/OVA NVs. As shown in Fig. 1A, PLGA/OVA NVs showed the particle size of ≈ 158 nm and the zeta potential of − 14.2 mV. After negatively charged RBC membrane coating, the particle size of MR-PLGA/OVA NVs increased to ≈ 173 nm and the zeta potential decreased to − 23.3 mV. TEM images also demonstrated that MR-PLGA/OVA NVs possessed the spherical core–shell morphology with the particle size of ≈ 160 nm, consisting with the DLS measurement (Fig. 1B). Besides, the particle size of MR-PLGA/OVA NVs had minimal alteration after incubation with serum or PBS during 7 days, suggesting that the NVs had ideal stability (Additional file 1: Fig. S1).
Alternatively, the SDS-PAGE demonstrated that MR-PLGA NPs had the similar protein expression as RBC membrane, which suggested that the RBC membrane was successfully coated onto PLGA NPs (Fig. 1C). After lyophilization and re-dissolution in PBS, the particle size of MR-PLGA/OVA NVs negligibly altered (Fig. 1D). The PLC and PLE of OVA was 8.1% and 75%, respectively. The in vitro OVA release from MR-PLGA/OVA NVs was further investigated. As shown in Fig. 1E, sustained OVA release was noted, achieving a cumulative amount of ~ 67.4% within 30 days.
Mannose-mediated BMDCs targeting and cellular uptake
Cellular uptake is the critical step for antigen delivery systems. CLSM images demonstrated that free FITC-OVA was negligibly taken up by BMDCs because of its hydrophilicity and high molecular weight (Fig. 2A). In comparison, green fluorescence was distributed in the cytoplasm when the FITC-OVA was loaded in MR-PLGA NVs, suggesting notable internalization of OVA assisted by MR-PLGA NVs. Pretreatment of BMDCs with mannose led to greatly reduced cytoplasmic distribution of green fluorescence, indicating mannose-mediated BMDCs targeting via recognition of mannose receptor. The cellular uptake level was further evaluated via spectrofluorimetry and flow cytometry (Fig. 2B, C). In consistence with CLSM observation, MR-PLGA/OVA NVs had higher cell uptake levels, while remarkable decrease of cellular uptake level was demonstrated under mannose pretreatment. These results collectively suggested that MR-PLGA/OVA NVs delivered OVA into BMDCs via mannose-mediated endocytosis.
In vitro MR-PLGA/OVA NVs-elicited BMDCs maturation
The process that NVs elicited antigen presentation and BMDCs maturation was then explored using flow cytometry and ELISA assay. Mature DCs overexpress the co-stimulatory molecules, MHC-I, MHC-II on the cell surfaces, including CD80 and CD86, along with cytokine production [45]. Moreover, SIINFEKL (OVA257-264 peptide) is able to complex with MHC-I for cross-priming CD8+ T cells. To explore the antigen presentation and BMDCs maturation, PBS, free OVA, PLGA/OVA NVs, R-PLGA/OVA NVs, MR-PLGA/OVA NVs, CMR-PLGA/OVA NVs were separately incubated with BMDCs for 12 h. Compared to free OVA or R-PLGA/OVA NVs, higher percentage of SIINFEKL+ CD11c+, MHC-II+ CD11c+ DCs were demonstrated after treatment with MR-PLGA/OVA NVs (Fig. 3A–D), indicating that mannose modification contributed to antigen presentation. Excitingly, CMR-PLGA/OVA NVs-treated BMDCs exhibited the maximum proportion of SIINFEKL+ CD11c+, CD80+ CD11c+, CD86+ CD11c+, MHC-II+ CD11c+, suggesting that CMR-PLGA/OVA NVs could effectively provoke antigen presentation and BMDCs maturation (Fig. 3A–H). In consistence with flow cytometry, the levels of cytokines in the supernatant collected from BMDCs were further revealed the similar results. IL-12 and TNF-α play an important role in stimulating T cell proliferation and eliciting the protective cellular immune responses [46,47,48]. As illustrated in Fig. 3I–K, significantly higher levels of IL-12, TNF-α, IFN-γ were found in BMDCs treated with CMR-PLGA/OVA NVs. These results collectively indicated that CMR-PLGA/OVA NVs could effectively provoke antigen presentation and the maturation of BMDCs in vitro.
In vivo biodistribution
To observe the in vivo distribution, the free Cy5.5-OVA, PLGA/Cy5.5-OVA NVs, R-PLGA/Cy5.5-OVA NVs and MR-PLGA/Cy5.5-OVA NVs were injected to mice. Compared to PLGA/Cy5.5-OVA NVs, the enhanced fluorescence signal of Cy5.5 was demonstrated in R-PLGA/Cy5.5-OVA NVs-treated mice (Fig. 4A, B), which was attributed to the splenic targeting ability of RBCs membrane. Excitingly, the Cy5.5 fluorescence in MR-PLGA/Cy5.5-OVA NVs-treated mice was stronger than that in other groups-treated mice at 72 h post injection. Moreover, the Cy5.5 fluorescence of MR-PLGA/Cy5.5-OVA NVs was still strong in the spleen even at 96 h post injection. The harvested tissues from immunized mice were further subjected to ex vivo imaging and quantitative analysis (Fig. 4B, C). Consistence with in vivo fluorescence imaging, the spleen accumulation level of Cy5.5 was higher than free Cy5.5-OVA and PLGA/Cy5.5-OVA NVs, which suggested that MR-PLGA/OVA NVs possessed the high spleen accumulation and retention.
In vivo CMR-PLGA/p54 NVs-elicited DCs maturation
To evaluate the protective immunity of NVs against virus infection in vivo, ASFV protein p54 was served as antigen to prepare the CMR-PLGA/p54 NVs (Additional file 1: Fig. S2). ASF is an acute, contagious, lethal infectious disease caused by ASFV, thus leading to the ~ 100% mortality rate and serious economic loss [49,50,51]. Therefore, it is imperative to develop vaccines to improve the protective immunity of animals against ASFV infection. DCs maturation has critical impact on eliciting the generation of antibody and T cell responses. To assess the DCs maturation in vivo, mice were immunized with different groups, and the DCs were further isolated from spleen on different time points (Fig. 5A). As shown in Fig. 5B–E, CMR-PLGA/p54 NVs induced higher level of stimulatory marker such as CD80 and CD86 compared with other groups, indicating that CMR-PLGA/p54 NVs could greatly promote splenic DCs maturation in vivo. Alternatively, the supernatants from splenic lysates were collected and measured by ELISA to investigate the level of cytokines including TNF-α, IL-12, IFN-γ. In consistence with the results of flow cytometry, the level of cytokines in CMR-PLGA/p54 NVs-treated spleen was higher than that in other groups-treated spleen (Fig. 5F–H, Additional file 1: Fig. S3). The similar results were also noted in LNs, wherein CMR-PLGA/p54 NVs significantly provoked the secretion of TNF-α, IL-12, IFN-γ (Additional file 1: Fig. S4). Collectively, these results indicated that CMR-PLGA NVs could dramatically promote DC maturation through CpG-assisted immune stimulation and mannose-assisted DC targeting.
In vivo CMR-PLGA/p54 NVs-elicited robust immune responses
Based on the remarkable ability of CMR-PLGA/p54 NVs to elicit DCs maturation, we further investigated the immunogenicity of CMR-PLGA/p54 NVs in vivo. Cellular immunity plays an important role on preventing viral infectious diseases. After being immunized with PBS, free p54, free p54 + Freund's adjuvant (FA), PLGA/p54 NVs, MR-PLGA/p54 NVs, and CMR-PLGA/p54 NVs on day 0, 14, 28, 35, spleen was harvested from immunized mice on day 49, homogenized to obtain single-cell suspensions. After being re-stimulated with CMR-PLGA/p54 NVs ex vivo, the proliferation proportion of splenocytes and the IFN-γ content was respectively 13.2-fold, 4.9-fold higher than that in free p54, suggesting that CMR-PLGA/p54 NVs could elicit antigen-specific cellular responses to achieve T cell proliferation (Fig. 6A, B). CD8+ T cells are one of critical immune cells that can protect body via killing the invading cells. Hence, the activation of CD8+ T cells was further assessed using flow cytometry. As illustrated in Fig. 6C and D, the proportion of CD3+ CD8+ T cells in mice immunized with PBS significantly increased from 4.24% to 17.95% in CMR-PLGA/p54 NVs immunized mice, which indicated that CMR-PLGA/p54 NVs had the potential to promote the proliferation and activation of CD8+ T cells. Alternatively, The CD4+ T cells have an important effect on regulating cellular and humoral immunity. After being immunized with CMR-PLGA/p54 NVs, the proportion of CD4+ T cells was significantly higher than other groups, indicating CMR-PLGA/p54 NVs-assisted proliferation and activation of CD4+ T cells (Fig. 6E, F). Collectively, CMR-PLGA/p54 NVs could activate both CD4+ and CD8+ T cell responses to elicit robust cellular immunity, which attributed to CMR-PLGA/p54 NVs-elevated spleen accumulation and targeting delivery antigen to DCs.
Alternatively, to investigate the humoral immunity, serum was collected from immunized mice for ELISA measurement. As shown in Fig. 6G, the elevated level of IgG in serum was observed when mice immunized with CMR-PLGA/p54 NVs. Alternatively, the ratio of IgG2a to IgG1 is a marker for Th1 and Th2 immune responses [52]. As shown in Fig. 6H, CMR-PLGA/p54 NVs could elicit higher IgG2a/IgG1 ratio compared with other groups, indicating that CMR-PLGA/p54 NVs favored a Th1-biased immune response. Besides, CMR-PLGA/p54 NVs could dramatically elicit the higher titers of p54-specific IgG than other groups, wherein the titers elevated by CMR-PLGA/p54 NVs were 17.3-fold higher than that induced by free p54 (Fig. 6I, J). The similar results were also obtained in p72 (ASFV antigen), where CMR-PLGA/p72 NVs significantly provoked the titers of p72-specific IgG in serum (Additional file 1: Fig. S5). Besides, B cell maturation and activation of germinal center (GC) in the spleen were further investigated. The GC response in the LNs or spleen is critical for long-lived humoral immunity [53, 54]. Compared with other control groups, higher percentage of CD19+ IgD+, CD21+ GC B cells was shown in the spleen collected from mice immunized with CMR-PLGA/p54 NVs, indicating that CMR-PLGA/p54 NVs also could promote B cell maturation (Fig. 6K, M). Collectively, these results suggested that the CMR-PLGA/p54 NVs could able to elicit robust humoral immune responses.
In vitro and in vivo safety
To evaluate the biocompatibility of MR-PLGA NPs, MR-PLGA NPs were incubated with BMDCs for 12 h. The cell viability remained more than 90% at the MR-PLGA NPs concentration up to 500 μg/mL, substantiating the good biocompatibility of CMR-PLGA NPs (Fig. 7A).
During the immunization period, the body weight of mice immunized with free p54 + FA began to decrease on day 28, and the nodules, hair loss were observed at the injection site. In comparison, NVs-immunized mice possessed the gradually increased body weight and minimal side effect (Additional file 1: Fig. S6). Histological examination measured by H&E staining further demonstrated that all NVs immunized mice had negligible damage of major organs (Additional file 1: Fig. S7). After 14 days post immunization of CMR-PLGA/p54 NVs, the organ coefficients (Fig. 7B), alanine creatinine (CRE), aminotransferase (ALT), urea nitrogen (UREA) and alkaline phosphatase (ALP) levels were not significantly increased (Fig. 7C). These results collectively indicated the in vivo desired safety of CMR-PLGA/p54 NVs.