Introduction

Vaccination plays a crucial role in controlling the dissemination of virus and reducing morbidity and mortality [1]. Subunit vaccines of recombinant proteins and epitope peptides are emerging in interest as a safer alternative to traditional vaccines [2,3,4]. However, the ability of these vaccines to elicit a long-lasting and potent immune response is limited because their drawbacks as comparatively lower molecular weights and weaker immunogenicity [5]. The need for adjuvants is urgent and increasing. Al, the most common adjuvant licensed for human use, has been widely employed for about 90 years [6, 7]. Its application is limited by disadvantages such as side effects and anergy to cellular immunity, which drives the development of new delivery system and adjuvants for these subunit vaccines [8, 9].

Biodegradable nanoparticles are being investigated as a delivery vector for vaccines and adjuvant systems in recent years that can boost weak antigen effectiveness by enhancing antigen processing and/or as immune-potentiating adjuvants to induce or potentiate immune responses. These nanoparticles are made of biodegradable materials such as polysaccharides, proteins, fatty acids lipids and polymers [10,4b). Within 24 h after OVA injection, pure antigen showed a rapid decline at the injection site, which easily entered the lymphatic circulation and quickly spread to the spleen, kidney, liver, and other major organs within 4 h (Fig. 4b, c). As time progressed the antigen distribution in major organs gradually decreased or even cleared. In contrast the OVA@DDAB/PLGA Nv groups showed the excellent ability of efficient antigen delivery to draining LNs. The radioactive signals of proximal LNs as inguinal LNs and popliteal LNs remained strong at 48 h post-injection (Fig. 4b). As time progressed the antigen mainly distributed into the spleen with trace found in the liver, kidneys. What is notable is that even 14 days post-injection antigens were still present in these locations. It was found that the OVA@DDAB/PLGA Nv at the injection site could be transported faster than OVA@Al, but slower than OVA (Fig. 4c). The OVA@DDAB/PLGA Nv not only formed a short-term "antigen repository" for antigen delivery, but also enhanced antigen transportation to LNs. Both of these effects are favorable for inducing an immune response. The Fig. 4d shows the diagram of internal organs in mice.

The biodistribution results indicated that the OVA@DDAB/PLGA Nv could effectively deliver antigen to secondary lymphoid organs such as the spleen and draining lymph nodes. Therefore, we further quantitatively studied the dynamic antigen biodistribution for different formulations in vivo. After intramuscular injection with 89Zr-OVA, 89Zr-OVA@Al or 89Zr-OVA@DDAB/PLGA Nv, the primary and secondary lymphoid tissues of mice were isolated at 0.5, 3, 6, 12, 24, 48, and 72 h to monitor radioactivity for calculating the biodistribution value (Fig. 5a; %ID/g: antigen uptake rate per gram of tissue at different times). The results showed that pure antigen (OVA) could be rapidly transported from the injection site to spleen with antigen uptake peaking at 6 h post injection (Fig. 5b, c). 89Zr-OVA@Al showed similar rapid antigen delivery to the spleen and peaking 6 h post injection as well (Fig. 5c). In contrast 89Zr-OVA@DDAB/PLGA Nv exhibited delayed antigen delivery to spleen with uptake peaking at 12 h post injection. A notable phenomenon is that 89Zr-OVA@DDAB/PLGA Nv showed two antigen transport peaks at 3 h and 12 h in draining LNs while 89Zr-OVA and 89Zr-OVA@Al only had a single peak at 12 h (Fig. 5d–f). Furthermore, antigen transported to the popliteal and inguinal LNs via the 89Zr-OVA@DDAB/PLGA Nv were maintained there for more than 72 h, while the antigen signal from 89Zr-OVA and 89Zr-OVA@Al dropped down to a very low level after 12 h (Fig. 5d, e). 89Zr-OVA@DDAB/PLGA Nv not only enhanced antigen delivery to proximal LNs, but also greatly promoted antigen transport to distal LNs such as cervical LNs (Fig. 5f). According these results, we hypothesize that 89Zr-OVA@DDAB/PLGA Nv transported antigen through two pathways: the first was direct transport into draining lymph nodes and the second was indirect transport by immune cells uptake such as DCs.

Fig. 5
figure 5

Dynamic antigen biodistribution of different vaccine formulations in mice. a Experimental design to evaluate the dynamic antigen biodistribution. b–f 89Zr-OVA biodistribution in injection site (b), spleen (c) Popliteal LNs (d), inguinal LNs (e), and cervical LNs (f). The primary organs and secondary lymphoid tissues of mice were isolated at 0.5, 3, 6, 12, 24, 48, and 72 h, and then radioactivity was quantified to calculate %ID/g value (% ID/g: antigen uptake rate per gram of tissue at different times). g immunohistochemical methods quantified the antigen in draining LNs (the brown areas represent the antigen OVA), and stained sections were measured by the automatic multispectral imaging system (PerkinElmer Vectra II), bar = 200 μm. Data are expressed as Mean ± STD (n = 6)

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OVA@DDAB/PLGA Nv Induced germinal centers formation in draining LNs and proliferative response of splenocyte

Antigen amounts in popliteal and inguinal LNs measured by flow cytometry also confirmed that OVA@DDAB/PLGA Nv promoted much more two folds antigen transport to lymph nodes as popliteal and inguinal LNs at 12 h than OVA and OVA@Al (Additional file 1: Fig. S5; P < 0.001). Immunohistochemical images suggested that the OVA@DDAB/PLGA Nv showed much higher ability in transferring antigen to LNs than OVA and OVA@Al (Fig. 5g and Additional file 1: Fig. S6). Moreover, antigens presented with the OVA@DDAB/PLGA Nv persisted in the LNs even seven days post immunization. These results implied that OVA@DDAB/PLGA Nv promoted both antigen migration into draining LNs at the early stage and subsequent continuous antigen stimulation to immune cells in LNs.

Maturation and activation of DCs is a prerequisite of antigen presentation, and directly affects the interaction with T cells [24, 28, 29]. The expression of co-stimulatory molecules CD86 on DCs was determined by flow cytometry. OVA@DDAB/PLGA Nv increased the expression of CD86 molecules on DCs in draining LNs (Additional file 1: Fig. S7a; NVs vs pure antigen: p < 0.0001). The expression of MHC I and MHC II on DCs from secondary LNs were also increased, which inducing by OVA@DDAB/PLGA Nv (Additional file 1: Fig. S6b, c). These results indicated that OVA@DDAB/PLGA Nv significantly promoted antigen cross-presentation. Expression of MHC II in the OVA@Al group gradually decreased after 24 h of immunization, while the MHC II in the OVA@DDAB/PLGA Nv group remained high (Additional file 1: Fig. S7c). These higher levels of MHC I and MHC II molecule expression on DCs suggested a stronger MHC-restricted antigen presenting pathway, which was favorable for T cell-mediated immunity. Therefore, these results indicated that the OVA@DDAB/PLGA Nv were superior to OVA@Al for inducing maturation and activation of DCs in secondary lymphocytes.

The OVA@DDAB/PLGA Nv showed great difference in antigen transportation from injection site to LNs and spleen compared to OVA and OVA@Al. Therefore, we further explored whether this difference would have a significant influence on subsequent immune activations. One of the most important functions of vaccines is to induce an effective specific and/or neutralizing antibodies against pathogens, which are mainly related to the level of follicular helper CD4+ T cells (Tfh) and formation of germinal centers represent B cells activation capacity. Flow cytometry was employed to detect the populations of follicular helper CD4+ T cells (CD4+ CXCR5hi PD-lhi) and germinal centers (GL-7hi B220+) in LNs. The OVA@DDAB/PLGA Nv significantly induced more cells populations of germinal centers in LNs over two-fold than OVA and OVA@Al (Fig. 6a and Additional file 1: Fig. S8b; for OVA and OVA@Al, P < 0.0001). And all three groups induced over 40% higher levels of follicular helper CD4+ T cells (Fig. 6b and Additional file 1: Fig. S8c). Immunohistochemical results showed that OVA@DDAB/PLGA Nv induced more germinal centers than the other two groups, notably 28 days post immunization (Fig. 6c and S9). Meanwhile, the proliferation and activation of lymphocytes in the spleen was important for immune system stimulation. The splenocyte from mice injected with OVA@DDAB/PLGA Nv showed much stronger proliferation ability than those with OVA (P < 0.0001) and OVA-Al (P < 0.01) (Fig. 6d-e). These results indicated that the OVA@DDAB/PLGA Nv could effectively promote germinal centers formation and splenocyte proliferation, which would be favorable for an effective immune response.

Fig. 6
figure 6

The OVA@DDAB/PLGA Nv induced the formation of germinal centers in draining LNs and proliferative response of splenocytes from immunized mice. a The number of germinal center (GL-7hi B220+ cells) in draining LNs. b The follicular helper CD4+ T cells (Tfh, CXCR5hi PD-1hi CD4+ T cells) in draining LNs. c Germinal centers in draining LNs determined by immunohistochemical staining; The circular brown signal is the germinal center B cell, and the dense circular positive area is the germinal center, which is circular or oval with a diameter of about 0.1 to 1.0 mm, and the bright area can be identified from the outside to the inside, bar = 1.0 mm. d OD450 nm value for immunized mice splenocytes stimulated with OVA in vitro were measured by CCK-8 kit; e Proliferation index of splenocytes. Data were expressed as means ± SEM (n = 6). (**p < 0.01; ***p < 0.001; ****p < 0.0001)

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OVA@DDAB/PLGA Nv induced the splenocyte activation and cytokines secretion

As a major indicator of lymphocyte activation in the spleen, the antigen specific CD4+ T, CD8+ T and B cells were evaluated and analyzed by flow cytometry after mice splenocytes were re-stimulated with antigen in vitro. CD69 and CD19 usually serve as the activation marker of T cells and B cells, which was used to measure lymphocyte activation. The mice were immunized three times and the spleen cells were isolated 35 days after the first immunized (Fig. 7a). The immunized splenocytes restimulated with OVA in vitro were employed for measuring CD4+ T cells (CD4+ CD69+ cells), CD8+ T cells (CD8+ CD69+ cells) and B cells (CD19+ CD69+ cells) by flow cytometry. The results suggested that the OVA@DDAB/PLGA Nv could promote more T and B cells activation than OVA and OVA@Al (Fig. 7b–d and Additional file 1: Fig. S10b–d; for CD4+ T cells comparing with OVA, p < 0.01; for CD4+ T cells comparing with OVA@Al, p < 0.05; for CD8+ T cells comparing with OVA, p < 0.001; for CD8+ T cells comparing with OVA@Al, p < 0.01; for CD19+ B cells comparing with OVA, p < 0.0001; for CD19+ B cells comparing with OVA@Al, p < 0.01). These results indicated that the OVA@DDAB/PLGA Nv could induce a strong activation of effector immune cells (T cells and B cells), which was necessary for enhancing system immune response.

Fig. 7
figure 7

OVA@DDAB/PLGA Nv induced splenocyte activation and cytokines secretion. a The schematic treatment schedule of the vaccine immunization. b, c, d The activation of CD4+ T cells (b) CD8+ T cells (c) and B cells (d) from mice after being immunized with different vaccine formulations were measured by flow cytometry. e, f, g, h The secretion of Th1-type cytokine of IFN-γ (e), TNF-α (f) and Th2-type cytokine of IL-6 (g), IL-4 (h) after intramuscular injection with different vaccines formulations were measured by an ELISA kit. i The ratio of IFN-γ to IL-4 secreted by splenocytes immunized with different vaccines. j Granzyme B secretion by splenocytes immunized with different vaccines were measured by an ELISA kit. Data was expressed as Mean ± STD (n = 6). (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

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It was reported that cytokines secretion from splenocytes play an essential role in provoking cell-mediated immune response [30]. Th1-type cytokines as IFN-γ, TNF-α and Th2-type cytokines including IL-4, IL-6 were evaluated. It was found that the OVA@DDAB/PLGA Nv significantly promoted more IFN-γ and TNF-α secretion than OVA (P < 0.0001) and OVA@Al (P < 0.01), and improved IL-6 and IL-4 secretion compared to OVA (P < 0.001) and OVA@Al (P < 0.05) (Fig. 7e–h). This implies that DDAB/PLGA nanoparticles also functioned as vaccine adjuvants by promoting Th1 and Th2 immune responses. The IFN-γ/IL-4 ratio was an indicator of the Th1/Th2 propensity of the immune response[31]. The ratio of IFN-γ/IL-4 induced by OVA@DDAB/PLGA Nv was higher than OVA (P < 0.01) and OVA@Al (P < 0.05), which further exhibited that the OVA@DDAB/PLGA Nv were more prone to induce Th1 type immune response (Fig. 7i). Meanwhile, the Nv induced higher levels of Granzyme B (Fig. 7j). The Granzyme B is an exogenous serine protease, which is derived from cytoplasm particles released by CTLs and natural killer cells (NK). Granzyme B plays an important role in cellular immune response, which can induce DNA degradation of target cells as infected cells and then lysis by activating the chain reaction of caspases. Therefore, our analysis indicated that the OVA@DDAB/PLGA Nv increased Th1 type cytokines secretion, which regulated the activation of immune cells and further induced the production of high levels of Granzyme B. In summary, the OVA@DDAB/PLGA Nv could effectively deliver antigens to LNs and spleen to induce a stronger immune response than OVA and OVA@Al, which demonstrated that the OVA@DDAB/PLGA Nv could induced not only cellular immunity from Th1 and Th2 responses, but also humoral immunity from B cells.

OVA@DDAB/PLGA Nv promoted T-cell-mediated immune response and elicited superior antibody response in mice

The ultimate goal of vaccination is to generate immune memory that can rapidly respond to pathogens upon reinfection, and memory T cells are an important components of these memory immune responses [32]. CD44hi CD62Llow and CD44hi CD62Lhi are regarded as markers for effector-memory T cells and central-memory T cells, respectively [32,33,34]. In our study, the OVA@DDAB/PLGA Nv generated significantly more effector T cells of CD4+ T and CD8+ T cells than OVA and OVA@Al (Fig. 8a, b and Additional file 1: Fig. S11b). Meanwhile, the OVA@DDAB/PLGA Nv also increased central-memory T cells of CD4+ T and CD8+ T cells which in theory will ensure a rapid immune response that will protect against reinfection (Fig. 8c, d and Additional file 1: Fig. S11b). In addition, cytotoxic T lymphocytes (CTL), a specific type of T cell, secretes a variety of cytokines and participates in the immune response. Its most important function is the specific killing effect on the pathogens that cause cellular infection [35]. CTL activation promotes the expression of CD107 molecules on the surface of CD8+ T cells [36]. Studies have found that CTL exerts its anti-killing effect mainly through two mechanisms: the first is the release of perforin and granzyme to kill target cells and the second is FasL-mediated cell apoptosis [37]. Analysis of the expression of CTL-related molecules by flow cytometry was an important indicator for evaluating immune response induced by vaccines. Meanwhile, the OVA@DDAB/PLGA Nv increased perforin release from CD8+ T cells compared to OVA (Fig. 8e and Additional file 1: Fig. S12b; P < 0.001). Compared to OVA, the OVA@DDAB/PLGA Nv could significantly up-regulate the CD107 and FasL expression on CD8+ T cells (Fig. 8f-g and Additional file 1: Fig. S12c, d; P < 0.01). These indicated that the OVA@DDAB/PLGA Nv could promote CTL activation and enhance CD107/FasL/perforin-mediated immune killing. In combination with the increasing expression of granzymes, we confirmed that the OVA@DDAB/PLGA Nv mainly relied on the mechanisms of Granzyme B and CD107/FasL/perforin expression to induce CTL responses.

Fig. 8
figure 8

Effects of different vaccines on memory T cell responses, CTL response, and antibody levels. a Effector memory (CD44hi CD62Llow) CD4+ T cells, b Cells of effector memory (CD44hi CD62Llow) CD8+ T cells, c Central memory (CD44hi CD62Lhi) CD4+ T cells, and D Central memory (CD44hi CD62Lhi) CD8+ T cells. The expression of Perforin (e), CD107 (f), and FasL (g) on CD8+ T cell in splenocytes. h Anti-OVA IgG titers in the serum from mice after being immunized with different vaccines for 35 days. i The composition ratio of IgG1 and IgG2c in serum from mice after immunized with different vaccines for 35 days. Data were analyzed by flow cytometry and expressed as Mean ± STD (n = 6). (*p < 0.05; **p < 0.01; ***p < 0.001)

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As the main immunoglobulin, Immunoglobulin G (IgG) is a key index to evaluate the level of immune response [38]. IgG1 and IgG2 are the two main subtypes of IgG. IgG2c/IgG1 can reflect the Th1/Th2 tendency in the immune response [32, 39, 40]. In this study, specific antibody titers in serum were detected by ELISA. We measured the antigen-specific IgG, IgG1, and IgG2c titers in serum from immunized mice with different vaccines. The results showed that OVA-specific antibody IgG gradually increased on day 14, 21, 28 and 35, and the IgG titers from OVA@Al and OVA@DDAB/PLGA Nv were much higher than OVA (P < 0.01), indicating that both mice groups exhibited stronger anti-OVA IgG responses (Fig. 8h). The OVA@DDAB/PLGA Nv induced higher IgG levels, while it induced stronger humoral immune response than OVA (for Day 21 and Day 35, P < 0.001; for Day 14 and Day 28, P < 0.01) (Fig. 8h), which suggested that the OVA@DDAB/PLGA Nv could induced humoral immunity. In addition, OVA@DDAB/PLGA Nv significantly increased much higher IgG2c proportion in the total IgG than OVA (P < 0.001) and OVA@Al (P < 0.01) (Fig. 8i), which indicated that the OVA@DDAB/PLGA Nv was capable of enhancing Th1 immune response (cellular immune response).

In addition to the immune effects, we also evaluated the safety of our OVA@DDAB/PLGA Nv delivery system. Blood samples were collected for the biochemistry of urea nitrogen (BUN), aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). Among these parameters, the values of BUN, AST and LDH were withing normal ranges for the OVA@DDAB/PLGA Nv group (Additional file 1: Fig. S13a, b, e). In contrast, ALT and ALP were found to be abnormally enhanced by Al adjuvant (Additional file 1: Fig. S13c-d), suggesting that this treatment had effectively spared the mice from hepatic or other organ damage. This indicates that its safer using DDAB/PLGA NP than Al adjuvant for immune protection and therapy.

Conclusion

In this paper, we successfully prepared OVA-loaded DDAB-PLGA nanoparticles (OVA@DDAB/PLGA Nv) with a positive charge. We explored the signal pathways that induced DCs activation and maturation in vitro and stimulated immune responses in vivo by the OVA@DDAB/PLGA Nv. Experiments performed in vitro show that the OVA@DDAB/PLGA Nv promoted the cellular uptake of antigen and the activation of BMDCs. The antigen uptake capacity of BMDCs incubated with OVA@DDAB/PLGA Nv was improved by more than twofold relative to pure OVA and OVA@Al at 12 h. Moreover, OVA@DDAB/PLGA Nv promoted antigen escape from the lysosomes into the cytoplasm, and induced ten times higher antigen cross-presentation activity compared to those OVA and OVA@Al. It was found that OVA@DDAB/PLGA Nv and OVA@Al exhibited different pathways to activate immunity. OVA@DDAB/PLGA Nv mainly activated DCs by p38 MAPK signal pathway. Meanwhile OVA@Al did not completely rely on the p38 signal pathway. Moreover, OVA@DDAB/PLGA Nv demonstrated two antigen transport pathways: one was direct rapid transport into draining lymph nodes at 3 h, and the other was an indirect transport pathway by DCs uptake at 12 h. These transport routes ensured that the OVA@DDAB/PLGA Nv could rapidly and continuously deliver antigen to the lymph nodes, which is favorable for stimulating an immune response in vivo. The antigen+ cells in inguinal LNs and popliteal LNs for OVA@DDAB/PLGA Nv were increased more than threefold compared to those of OVA@Al and OVA at 24 h. Furthermore, the antigen of OVA@DDAB/PLGA Nv persisted in LNs for more than 7 days, which induced germinal center formation as two times higher than those of OVA@Al and OVA. After immunization, OVA@DDAB/PLGA Nv induced comparable anti-OVA IgG and much higher ratio of IgG2c/IgG1 compared to OVA@Al. It also effectively activated CD4+T, CD8+T and B cells that further induced immune memory. As expected, OVA@DDAB/PLGA Nv elicited a strong cytotoxic T lymphocytes (CTLs) response. Stimulation with OVA@DDAB/PLGA Nv increased the proportion of CD107+CD8+, Fasl+CD8+ and perforin+CD8+ T cells and the secretion of IFN-γ and Granzyme B. These results indicate that the DDAB/PLGA NP was a potent platform to improve vaccine immunogenicity by novel signaling pathway in DCs, rapid and massive transport antigens to LNs.