Evaluation of Different Types of Adjuvants in a Malaria Transmission-blocking Vaccine

Abstract

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA+QS-21 and QS-21+CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA+QS-21, QS-21+CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21+CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

Graphical Abstract

1. Introduction

The Plasmodium parasite is responsible for the infectious disease malaria, which poses a significant health risk to humans. Vaccines might serve as an integral strategy for the elimination of malaria in endemic areas and complement other interventions such as drug therapies and mosquito abatement. The progress of malaria vaccine development over the past decades has been hindered by significant challenges, including the inability to confer long-term protective immunity and generate effective cellular immune responses. The most advanced vaccine RTS, S/AS01 shows only modest efficacy in children and infants [1]. Vaccine efficacy declines with time and falls short of the standard set by the Malaria Vaccination Technology Roadmap [2]. The decreased efficacy is associated with diminished levels of antibodies recognizing the RTS,S/AS01 vaccine target, the circumsporozoite protein (CSP), suggesting that protection relies on the maintenance of consistently high circulating antibody levels [3]. The enhancement of antigen immunogenicity and maintenance of elicited antibody levels constitute a crucial strategy for optimizing the development of malaria vaccines. Thus, novel approaches are desired to improve the malaria vaccine design.

The use of adjuvants is widely acknowledged to not only enhance the strength and longevity of the antibody response, but also exert potential influence on the appropriate type of immune response [4]. Adjuvant incorporation into a vaccine could reduce the antigen dosage and the number of immunizations that are needed to elicit a protective immune response. Thus, the selection of appropriate adjuvants impacts the efficacy of a vaccine. Generally, adjuvants can be categorized as immunostimulants and delivery systems, depending on their mechanisms of action and effects on the immune system [5]. Immunostimulants typically induce the maturation and activation of antigen-presenting cells (APCs) by targeting toll-like receptors (TLRs) and other pattern recognition receptors (PRRs). Delivery systems serve as carrier materials that enhance antigen presentation by extending the bioavailability of loaded antigens and directing them toward lymph nodes or APCs. The mechanisms of action and immunological properties of adjuvants have been systematically reviewed [6, 7].

The adjuvants evaluated to date encompass a wide range of compound classes, including mineral salts, emulsions, saponins, nanoparticles, polymers, liposomes, and synthetic small molecular agonists [7]. The first adjuvant licensed for human use was aluminum salts, which have been extensively utilized in human vaccines against various infectious diseases, including dengue, tetanus, and hepatitis B [8]. It has been demonstrated that the adsorption of antigens onto aluminum hydroxide (Alum) could maintain a high concentration of antigens, thereby enhancing antigen uptake by APCs and further bolstering immune responses [9]. A substantial number of mechanistic studies suggest that Alum promotes strong Th2-biased immune responses and enhances IgG1 levels [10].

TLRs are a group of PRRs that recognize microbial pathogens to initiate responses to infection [11]. To date, TLR agonist-based adjuvants such as monophosphoryl lipid A (Salmonella minnesota R595) (MPLA) and CpG ODN (cytosine phosphoguanosine oligodeoxynucleotides) have been formulated in licensed vaccines for their adjuvant activity. MPLA is a lipopolysaccharide-based TLR-4 agonist [12, 13]. AS01, a liposome-based vehicle containing MPLA, was the first malaria vaccine adjuvant to undergo pilot implementation [14]. Activation of TLR-4 by MPLA leads to the production of proinflammatory cytokines, including TNF-α and IL-6, further enhancing the adaptive immune response [15]. MPLA can induce a Th1-type immune response by promoting IFN-γ production [16]. CpG ODN 1826 is a TLR-9 agonist that could augment the antibody response and trigger Th1 immunity [17]. Additionally, it facilitates the production of proinflammatory cytokines, such as IL-6, IL-12, and IFN-γ. It has been demonstrated that CpG ODN could activate dendritic cell (DC) maturation into specialized APCs and accelerate antibody synthesis for protective immunity [18]. Saponins, which are natural glycoside compounds, represent another category of adjuvants. QS-21 is a saponin-like adjuvant that could enhance both Th1- and Th2-type immune responses, leading to increased levels of IgG2a, IgG2b, and IgG1 antibodies, as well as antigen-specific cytotoxic T cells [19]. The current usage of QS-21 as an adjuvant in vaccines against infectious diseases is widespread, and it is also employed as a component of the AS01B adjuvant for vaccines for human use [6]. Attempts are underway for cancer vaccines to create new optimal QS-21 adjuvant combinations with various adjuvants, such as MPL and CpG ODN [20, 21].

To disrupt malaria transmission in epidemic regions, TBVs aim to reduce or interrupt the transmission of malaria in vectors and human hosts by targeting sexual stage and mosquito stage development [22]. Pfs30, Pfs48/45 and Pfs47, Pfs25 and PfHAP2 are lead TBV candidates, some of which are in clinical development [22]. In addition, one candidate TBV antigen is the ookinete surface protein PSOP25, which has been demonstrated to be crucial for male gametocyte exflagellation and ookinete development in the rodent malaria parasite, Plasmodium berghei [23, 24]. Antibodies against PSOP25 effectively suppress mosquito midgut oocyst production [23]. In this study, we compared and determined the efficacy of the PSOP25 protein in eliciting antibody and immune responses with three vaccine adjuvants approved for human use: MPLA, CpG ODN 1826 (CpG for short), and QS-21 [6, 19, 25]. We also evaluated the effectiveness of two combination adjuvants: MPLA+QS-21 and QS-21+CpG. The transmission-blocking efficacy of sera induced by these vaccines was assessed using in vitro and in vivo assays. This study seeks to contribute to the development of adjuvant-assisted malaria subunit vaccines.

2. Methods

2.1. Mice, Parasites and Mosquitoes

Six- to eight-week-old female BALB/c mice were purchased from the Beijing Animal Institute (Beijing, China) and housed at the central animal facilities of China Medical University. The mice were maintained under “specific pathogen-free” conditions per the guidelines established by China Medical University animal facilities central (Shenyang, China). Animal use was carried out according to the guidelines of the animal ethics committee of China Medical University. The license number for this study is CMU2022108.

The P. berghei ANKA 2.34 malaria parasite strain was maintained by serial passages and used for challenge infections or ookinete culture. Adult female Anopheles stephensi mosquitoes (Hor strain) were kept as described [23].

2.2. Expression and purification of recombinant PSOP25 protein

A DNA fragment encoding amino acids 45–245 of PSOP25 (PBANKA_111920) was cloned into the prokaryotic expression vector pET32a (+) (Novagen, USA). The recombinant plasmid was transformed into Escherichia coli (E. coli) RGB and the Histagged recombinate protein expression was induced with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) (Sigma, USA) at 19°C for 8 h. The bacteria were pelleted by centrifugation, resuspended in binding buffer (10 mM imidazole (Geneview, USA), 300 mM NaCl, and 50 mM sodium phosphate; pH 8.0), and lysed by freeze-thawing twice and sonication. The soluble rPSOP25 was bound to nickel-agarose gel 6FF at ambient temperature for one hour, washed, and eluted with His natural buffer containing varying concentrations of imidazole. Deep desalination of the final purified rPSOP25 was performed in 0.1 M phosphate-buffered saline (PBS) overnight at 4°C, and a sample of the protein was then separated by SDS-PAGE for validation. The endotoxin from the recombinant protein was removed by ToxinEraser^™ kit (Genscript, China), according to the manufacturer’s protocol.

2.3. Mouse immunization procedure

The adjuvants used in this study included MPLA (S. minnesota R595) (InvivoGen, USA), CpG-ODN 1826 (InvivoGen, USA), QS-21 (Alpha Diagnostic International, USA), and Alum (Thermo Fisher, USA). The study involved eight groups of mice, with each group consisting of twelve individuals. On days 0, 14, and 28, the mice were subcutaneously vaccinated with the prime and booster doses of rPSOP25 at 50 μg and 25 μg, respectively, along with various adjuvants, as shown in Table 1. The dosage of the adjuvants was determined based on the manufacturers’ protocols, adhering to the recommended maximum amount. For the groups receiving a combination of adjuvants, each component was administered at half the standard dosage. Blood samples were collected from tail vein at 14, 28, 38, 112, and 180 days after the prime vaccination. The blood samples were centrifuged at 13800 g for 15 minutes to isolate the serum and stored at −80°C until further use. At day 38 after initial immunization, three mice were anesthetized with isoflurane and euthanized per group, then spleens were harvested.

Table 1.

Vaccine dosages and vaccination schedules

Group	Adjuvant dosage	Protein dosage (prime)	Protein dosage (boost)
PBS
rPSOP25		50 μg	25 μg
rPSO25/MPLA	20 μg	50 μg	25 μg
rPSOP25/CpG	50 μg	50 μg	25 μg
rPSOP25/QS-21	20 μg	50 μg	25 μg
rPSOP25/MPLA+QS-21	10 μg+10 μg	50 μg	25 μg
rPSOP25/QS-21+CpG	10 μg+25 μg	50 μg	25 μg
rPSOP25/Alum	100 μL	50 μg	25 μg

2.4. Detection of serum IgG, IgG1, and IgG2a levels

IgG, IgG1, and IgG2a levels were measured by enzyme-linked immunosorbent assay (ELISA) with five separate collections of serum. The 96-well ELISA plates were incubated overnight with carbonate buffer containing rPSOP25 protein (10 μg/mL) at room temperature. The plates were blocked for 1 h at 37°C with 1% bovine serum albumin (BSA) (Sigma, USA) diluted in PBS. Then 100 μL of antiserum (1:2000 to 1:256,000) from three immunizations diluted in blocking solution was added to each well and incubated for 2 h at 37°C. HRP-labeled sheep anti-mouse IgG (1:10,000) (Thermo Fisher, USA), or IgG1 and IgG2a (1:2000) (Abclone, China) were applied to appropriate wells and incubated for 1 h at 37°C. After washing with PBS/T, 100 μL of HRP-labelled TMB substrate solution (Beyotime, China) was added to each well and incubated at RT for 10 min. The reactions were terminated by adding 100 μL of termination solution (without sulfuric acid) (Beyotime, China) to each well, and the optical density (OD) was immediately determined at 450 nm using a microplate reader (ELx808, BioTek). The serum IgG endpoint titers were defined as the highest antiserum dilution at which the OD value at 450 nm exceeded the mean of the PBS control+3×SEM.

2.5. Avidity of serum IgG measured by ELISA

ELISA plates were incubated with carbonate buffer (10 μg/mL) containing rPSOP25 overnight at room temperature. Each set of wells was done in duplicate. The plates were blocked with 1% BSA for 1 h at 37°C, and then 100 μL of antiserum obtained from the third immunization (1:200) was diluted with the blocking solution and added to each well and incubated at 37°C for 2 h. Then 100 μL of PBS and 100 μL of 8 M urea (Solarbio, China) was added to each well, and incubated for 45 min at room temperature. HRP-labeled sheep anti-mouse IgG (1:10,000) was then added to each well, followed by HRP-labelled TMB chromogenic solution and a 15 min incubation. The reactions were terminated using a termination solution without sulfuric acid, and the OD values were observed at 450 nm with a microplate reader (ELx808, BioTek).

2.6. IFA

The ability of antiserum collected from each immunization group to recognize ookinete native parasite PSOP25 antigen was confirmed using IFA. The parasites were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde, then permeabilized with 0.1% Triton X-100. After washing in PBS, the samples were blocked with a blocking buffer (3% BSA in PBS) at RT for 1 h, and then were incubated with antisera (1:200) from each group. PBS was used as the negative control, and all samples were co-incubated with rabbit antisera recognizing the ookinete surface protein Pbs25 (1:500) at 37°C for 1 h as a marker for ookinetes. The samples were then incubated at RT for 1 h with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (1:500, Invitrogen) and Alexa Fluor 555-conjugated goat polyclonal antibody to rabbit IgG (1:500, Abcam). Cell nuclei were counterstained with Hoechst 33258 solution (1:1000, Sigma). All samples were imaged and analyzed using a fluorescence confocal laser scanning microscope (STELLARIS 5, Leica Microsystems).

2.7. Cytokine analysis

BALB/c mice from all groups were anesthetized by isoflurane and then sacrificed. Supernatants of splenocytes were collected from each immunization group (n=3), and the expression levels of TNF-α, IFN-γ, IL-21, IL-4, IL-6, and IL-10 were determined using an Immune Factor Assay Kit (R&D, USA), according to the manufacturer’s protocol. OD values were measured at 450 nm with a microplate reader (ELx808, BioTek). The cytokine expression levels in the samples were determined based on standard curves generated for each cytokine.

2.8. Flow cytometry analysis

BALB/c mice were sacrificed on day 38 post-first immunization. Spleens from each group were collected 10 days after the third immunization. Splenocyte suspensions were homogenized using a sterile sieve and then washed with PBS. Erythrocytes were lysed with 0.17 M NH₄Cl and washed twice with RPMI-1640. A splenocyte suspension was then prepared using RPMI-1640 containing 10% FBS, with an adjustment of the cell concentration to 1×10⁷ cells/mL. Splenocyte subsets were defined as: DC (CD11c⁺), mature DC (CD11c⁺MHCII⁺), convential dendritic cell (cDC) (CD11c⁺CD11b⁺B220⁻), plasmacytoid dendritic cell (pDC) (CD11C⁺CD11b⁻B220⁺), T helper 1 cell (Th1) (CD4⁺IFN-γ⁺), memory T cell (CD4⁺CD62⁺CD44⁺), follicular helper T cell (Tfh) (CD4⁺CXCR5⁺PD-1⁺), GC B cell (B220⁺CD95⁺GL7⁺), and plasma cell (B220⁺CD138⁺). To assess the function of DCs, BV605-conjugated anti-CD11c mAb (eBiosciences, USA), APC-conjugated anti-MHCII mAb (eBiosciences, USA), FITC-conjugated anti-CD11b mAb (Biolegend, USA), and PE-Cy7-conjugated anti-B220 mAb (eBioscience, USA) were used for staining. To investigate Th1 cells, APC-conjugated anti-IL-4 mAb (BD Bioscience, USA) and BV650-conjugated anti-IFN-γ mAb (Biolegend, USA) were used for immunostaining. For the analysis of memory T cells, FITC-conjugated anti-CD44 mAb (BD Bioscience, USA) and APC-Cy7-conjugated anti-CD62L (BD Bioscience, USA) were utilized for staining. For the study of Tfh cells and GC B cells function, staining was conducted with PE-conjugated anti-CXCR5 mAb (Biolegend, USA), BV421-conjugated anti-PD-1 mAb (BioLegend, USA), APC-conjugated-anti-CD95 mAb (Biolegend, USA), and PE-conjugated anti-GL7 mAb (Biolegend, USA). To investigate PC, PE-Cy7 conjugated anti-B220 mAb (eBioscience, USA) and BV785 conjugated anti-CD138 mAb (BioLegend, USA) were used for staining. The samples were incubated with corresponding fluorescent antibodies at 4°C for 30 min, protected from light. After centrifugation at 500 g for 5 min, the supernatant was discarded and the precipitate was resuspended in 200 μL PBS for flow cytometry analysis using a FACS Celesta (FACScan, BD) and FlowJo software (version 10.6).

2.9. In vivo and in vitro transmission-blocking activity

On day 14 after the third immunization, mice from each immunization group were infected intravenously with 5 × 10⁶ P. berghei parasites. After 3 days, 10 μL of blood was mixed with 90 μL of ookinete medium (RPMI 1640, 50 mg/L penicillin, 50 mg/L streptomycin, 100 mg/L neomycin, 25% [v/v] heat-inactivated fetal calf serum, 6 U/mL heparin, pH=8.0) and incubated at 19°C for 24 h. Then 0.5 μL of the culture was utilized to prepare smears, which were subsequently fixed and labeled with anti-Pbs25 mAb (1:500) as an ookinete marker, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500). The number of ookinetes per well (equivalent to 0.5 μl of cultures) was determined using a fluorescence microscope (Olympus, U-HGLGPS). The mice were then euthanized.

For the in vitro assay, the naïve mice were intravenously injected with 5 × 10⁶ P. berghei parasites. On day 3 post-infection, the parasitemia of infected red blood cells was determined, and the exflagellation of male gametocytes was assayed. Then 10 μl of infected blood was taken from each mouse and added to 90 μl ookinete transformation medium containing the serum from each group at final dilutions of 1:5 and 1:10. After incubation for 24 h at 19°C the number of ookinetes was calculated as described above.

For the in vivo transmission-blocking assays, mice were injected via the tail vein with 5×10⁶ P. berghei parasites. Three days after infection, blood was collected from the tail vein and examined for gamete exflagellation. Antisera (100 μL) collected from mice from different immunization groups were passively transferred to infected mice via the tail vein. One hour following the serum transfer, mice were anesthetized by isoflurane and exposed for 2 h to at least 50 pre-starved female Anopheles mosquitoes. After removing the unfed mosquitoes, the engorged mosquitoes were maintained for 10 days and dissected to determine midgut infection. Midguts were stained with 0.5% mercurochrome, and oocysts were counted to assess transmission reduction activity (TRA) (the % inhibition in the mean oocyst count per mosquito) and transmission blocking activity (TBA) (the % inhibition in the prevalence of infected mosquitoes). As per Animal ethics guidelines, humane endpoints were considered in all the in vivo experiments.

2.10. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 10.1.2 software and SPSS software, version 23.0. One-way ANOVA was used to compare the IgG subclasses, cytokines, ookinete numbers in vivo, and the differences between immune cells among vaccination groups. To account for multiple comparisons following ANOVA, the Tukey’s multiple comparisons test was employed for comparison among groups. Two-way ANOVA was used to compare antibody levels, long term comparison and ookinete numbers in vitro among the vaccine groups, the Tukey post hoc test was employed for inter-group comparison. The oocyst density was analyzed by the Kruskal-Wallis H test, Bonferroni post hoc test was used for multiple comparsions. Illustrations were created with figdraw (https://www.figdraw.com/) and biorender (BioRender.com).

3. RESULTS

3.1. Determination of antibody levels and avidity

The 45–245 aa region of the PSOP25 antigen was selected for this study due to its predicted abundance of B-cell epitopes, using the ABCpred website (http://webs.iiitd.edu.in/raghava/abcpred/) (Supplementary Figure S1A). To express and purify the recombinant PSOP25 protein (rPSOP25), the 45–245 aa fragment was expressed in the E. coli RGB/pET32a expression system (Supplementary Figure S1B), and a protein band of approximately 42 kDa was detected by SDS-PAGE (Supplementary Figure S1C). To evaluate potential adjuvant activities, BALB/c mice were immunized subcutaneously with rPSOP25 formulated with different adjuvants on days 0, 14, and 28, as indicated in the vaccination strategy (Figure 1A). Sera were collected from each group to determine the antibody levels by ELISA. The results showed that antibody levels after the 3^rd immunization were significantly higher than the 2^nd immunization in all groups containing the rPSOP25 antigen (P < 0.0001) (Figure 1B). Animals immunized with adjuvant-formulated vaccines exhibited a robust antibody response, as indicated by the anti-PSOP25 IgG antibody titer reaching 1:256,000 after booster immunization, which was higher than rPSOP25 (1:128,000) groups (Figure 1C). These data confirmed that the immunogenicity of rPSOP25 could be enhanced through administration with extrinsic adjuvants.

Figure 1. Antibody response in sera from vaccinated mice at different time points.

(A). Schematic representation of animal vaccination and detection strategies (n=12). (B). Anti-rPSOP25 IgG levels were analyzed after three immunizations . **** indicates a significant difference at P < 0.0001 (Two-way ANOVA, Tukey post hoc test). (C). Specific IgG titers were analyzed on the third immunization. The dashed line (cut-off value) indicates the mean of PBS antiserum +3×SEM. (D). Antibody avidity was assessed on day 38 in serum samples from mice vaccinated with the rPSOP25 vaccines. Avidity index values were determined by measuring the resistance of antibody-antigen complexes to 8 M urea. The avidity index was calculated as the ratio of urea-treated wells’ mean ELISA OD450 value to PBS control wells multiplied by 100. Data were representative of three independent experiments. Error bars were indicated as mean ± SD. *** indicates a significant difference at P < 0.001, **** indicates a significant difference at P < 0.0001 (One-way ANOVA, Tukey post hoc test).

The antibody avidity index plays a crucial role in predicting the efficacy of an antibody and represents the strength of the binding between the antibody and its target cluster of antigenic determinants [26]. We evaluated the antibody avidity on 38 days following the initial immunization (Figure 1D). The results demonstrated that all adjuvant groups showed significantly higher antibody avidity indexes than the rPSOP25 antigen alone (P < 0.001), while there were no significant differences between the adjuvant groups (P > 0.05). These findings showed that the anti-rPSOP25 antibody level and avidity can be increased using the evaluated adjuvants.

3.2. The level of IgG subclasses in adjuvanted vaccines

Generally, a Th2-type response favors the production of IgG1 antibodies, while the presence of IgG2a indicates a bias towards a Th1 response. To determine the immune response induced by different adjuvants, we evaluated the IgG1 and IgG2a levels by ELISA for immune sera collected on days 14, 28, and 38 following the first immunization (Figure 2). The results revealed that the rPSOP25 antigen alone displayed higher levels of IgG1 than IgG2a at all time points, indicating that it primarily elicited Th2-biased immune responses. At day 14 post-initial immunization, the highest level of anti-rPSOP25 IgG1 was detected in the QS-21 group (Figure 2A). The IgG1 levels of all tested adjuvant groups were significantly higher than the rPSOP25 antigen alone on day 28, and there was no difference among the adjuvant groups (Figure 2B). Following the second boost immunization, all adjuvant groups had elevated IgG1 levels compared with the rPSOP25 group. The QS-21, MPLA+QS-21, and QS-21+CpG groups induced higher IgG1 levels than the Alum group (Figure 2C). Only the QS-21 and QS-21+CpG adjuvant groups induced an IgG2a response at 14 days after the prime vaccination (Figure 2D). In line with expectations, the Alum adjuvant barely increased IgG2a antibodies, even after boosting. The level of IgG2a induced by QS-21+CpG was higher than the other adjuvanted vaccine groups (Figures 2E–F), which may be related to the combined effect of QS-21 and CpG.

Figure 2. Analysis of antibody subclasses in serum samples from mice vaccinated with the rPSOP25 vaccines.

The IgG1 (A-C) and IgG2a (D-F) levels were detected in mice serum at days 14, 28, and 38 after the primary immunization. The dashed line (cut-off value) indicates the mean of PBS antiserum+3 × SEM. (G). The IgG1/IgG2a ratios were calculated for each mouse from three immunizations. The dashed line indicates IgG1/IgG2a ratio = 1. Results are shown as mean±SD of OD calculated from three to six mice in each group.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test).

To elucidate the type of induced immune responses in mice, we compared the IgG1/IgG2a ratio among the vaccine groups. At day 14, the rPSOP25 antigen alone showed the highest and most significant IgG1/IgG2a ratio of 6.5, followed by the groups receiving rPSOP25 formulated with MPLA+QS-21 (3.1) and MPLA (3.0). The lowest IgG1/IgG2a ratio was observed in the QS-21+CpG group (1.3). The IgG1/IgG2a ratio in all adjuvant vaccine groups ranged from 1.8 to 2.0 on days 28 and 38, and no statistically significant difference was observed when compared with the rPSOP25 antigen alone (1.7) (Figure 2G). These results indicated that the adjuvants tested here did not alter the type of immune response compared to the antigen alone.

3.3. Validation of the elicited specific anti-PSOP25 antibodies

PSOP25 is predominantly expressed on the surface of ookinetes [23]. The ability of the antibodies induced by the vaccines to recognize the ookinete native surface antigen was determined by indirect immunofluorescence assays (IFA) (Supplementary Figure S2). Sera from all vaccination groups, including the rPSOP25-only group, recognized the ookinete surface of P. berghei parasites versus no reactivity observed with serum from PBS-injected control mice. This data indicated that rPSOP25 combined with all adjuvants could elicit antibodies capable of binding the native structure of the PSOP25 protein.

3.4. Determination of ookinete formation and transmission-blocking activity

To assess the ability of the antisera to inhibit ookinete transformation, mice were intravenously infected with 5◊10⁶ P. berghei parasites at day 14 after the 3^rd vaccination. Three days later, blood was collected, and the in vitro formation of ookinetes was quantified (Figure 3A). As expected, the PBS group had the highest number of ookinetes, followed by the rPSOP25 group alone. The adjuvant groups showed different capacities to inhibit ookinete formation compared to the PBS control group. The serum from the rPSOP25 group exhibited minimal impact on ookinete formation, resulting in a 30% reduction in ookinetes. The single adjuvant groups exhibited mild decreases in ookinete numbers, with the rPSOP25 formulated with the MPLA and QS-21 showing reductions of 40% and 46%, respectively. The inhibition effect on ookinete transformation of CpG group was comparable with Alum group , which resulted in a 75% reduction in ookinete numbers. The combination adjuvant groups, MPLA+QS-21 (P < 0.01) and QS-21+CpG (P < 0.0001), also exhibited a significant reduction in ookinete formation compared to the antigen-only group.

Figure 3. Transmission-blocking activities of the rPSOP25 vaccine assessed in vitro and in vivo.

(A). Inhibition of ookinete formation by the different vaccines. Immunized mice (n= 3) were intravenously.challenged with P. berghei parasites on day 14 after the third immunization. Data were representative of three independent experiments.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test). (B). Effects of the immune serum on ookinete numbers in vitro. The antisera were used at dilutions of 1:5 and 1:10. Means were calculated from three independent experiments. Data were representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Two-way ANOVA, Tukey post hoc test). (C). Direct mosquito feeding assay. Individual data points represent the number of oocysts found in mosquitoes 10 days post-feeding. Horizontal bars indicate the mean number of oocysts per midgut and error bars indicate SD within individual treatments. Data were representative of three independent experiments.* P < 0.05, *** P < 0.001, **** P < 0.0001 (Kruskal-Wallis H, Bonferroni post hoc test).

In the second approach, ookinete cultures were established using gametocytes from a naïve P. berghei-infected mouse, supplemented with 1:5 or 1:10 diluted pooled immune sera derived from the vaccine groups. The antisera collected from the antigen-only group exhibited a minimal reduction in the ookinete numbers, with decreases of 21% and 12% observed at dilutions of 1:5 and 1:10, respectively. Among the adjuvant vaccine groups, rPSOP25 formulated with MPLA consistently showed a weak effect inhibiting ookinete formation, compared with the antigen-only group at 1:5 and 1:10 dilutions. The antiserum from the CpG group or its combination group (QS-21+CpG) exhibited robust reductions in ookinete formation at both 1:5 (reduced by 67% and 66%, respectively) and 1:10 dilution (reduced by 56% and 63%, respectively), surpassing even the Alum group (Figure 3B).

Next, we used direct mosquito feeding assays (DFA) to evaluate the transmission reducing activity (TRA) and transmission-blocking activity (TBA) of antisera elicited by antigen formulated with CpG and QS-21+CpG. Mice infected with P. berghei parasites were passively transferred with different antisera through the tail vein. At 10 days after feeding, about 30 mosquitoes were dissected from each group to assess oocyst density and the prevalence of infection. In mosquitoes fed on mice receiving sera, the mean number of oocysts per midgut and infection prevalence from the rPSOP25-only group were reduced by 30% and 10%, respectively (Figure 3C, Table 2). As anticipated, the CpG group and QS-21+CpG group exhibited a significant reduction in both mean oocyst density (74% and 71%) and infection prevalence (30% and 24%) compared to the PBS control group. The results suggested that the QS-21+CpG combined adjuvant did not exert synergistic benefits. The CpG vaccine group showed higher TRA and TBA when compared with the Alum group (Figure 3C, Table 2). These findings suggested that rPSOP25 formulated CpG elicited antisera with potential improvement in both TRA and TBA.

Table 2.

Evaluation of antiserum TBA by mosquito feeding assays

Group	Oocyst density mean (range) (n = 30)	TRAa (%)	Prevalence of infection mean (n=30)	TBAb (%)
PBS	96.1 (0–237)		96.8 (29/30)
rPSOP25	67.3 (0–117)	30.0	86.7 (26/30)	10.1
CpG	25.3 (0–90)	73.7	66.7 (20/30)	30.1
QS-21+CpG	28.0 (0–90)	70.9	73.3 (22/30)	23.5
Alum	34.2 (0–101)	64.4	73.3 (22/30)	23.5

^aTRA (%) was calculated as (mean oocyst density _PBS – mean oocyst density _{PSOP25/CpG/QS-21+CpG/Alum}) / mean oocyst _PBS × 100%

^bTBA (%) was calculated as %prevalence _PBS – % _{prevalencePSOP25/CpG/QS-21+CpG/Alum}

3.5. Cytokine response in vaccinated mice

To elucidate the cytokine responses elicited by rPSOP25 adjuvanted vaccines, we analyzed the cytokine levels for IFN-γ, TNF-α, IL-6, IL-21, IL-4, and IL-10 within culture medium of splenocytes that were harvested on day 38 from mice of each vaccination group. IFN-γ and TNF-α (cellular immunity index) and IL-4 and IL-6 (humoral immunity index) were used as indicators of the Th1 and Th2 type of immune responses, IL-21 (humoral immunity index) were used as indicators of Tfh response, respectively, while IL-10 was used to assess the overall regulation of the immune system [27].

The ELISA results showed that the cytokine levels of IFN-γ, TNF-α, and IL-6 in the CpG group were considerably increased compared to the rPSOP25 group (Figure 4). In addition, the MPLA, MPLA+QS-21, and QS-21+CpG groups had significantly increased levels of IFN-γ (Figure 4A) and IL-6 (Figure 4C).in immunized mice. The QS-21 group had increased levels of IFN-γ and TNF-α in comparision to the rPSOP25 group (Figure 4A–B). All tested adjuvants could initiate Th1 responses, as evidenced by increased IFN-γ production in splenocytes (Figure 4A). The levels of Th2-related cytokines IL-4 and IL-10 did not show a statistically significant difference between the group exposed to antigen alone and the adjuvanted vaccines (P > 0.05). (Figure 4D–E). The level of IL-21 was below the limit of detection; therefore, the data were not presented. The effect of cytokines on the immune response may be dynamic and multifaceted, so further study is needed to illustrate the time course of cytokines.

Figure 4. Cytokine levels induced by immunization with rPSOP25-based vaccines.

Spleen cells were collected from immunized mice after the third immunization. The secreted cytokines IFN-γ (A), TNF-α (B), IL-6 (C), IL-4 (D), and IL-10 (E) were measured from the supernatants using mouse uncoated ELISA kits. Shown are means with SD of three mice per group. Data were representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test).

3.6. The immune response elicited by different adjuvanted vaccines

To clarify the innate immune response following immunization with rPSOP25 formulated with different adjuvants, we evaluated in the vaccinated mice the DC subtypes known as cDC and pDC. The CpG and Alum adjuvants significantly increased the levels of both cDC (CD11c⁺CD11b⁺) and pDC (CD11c⁺B220⁺) compared to the antigen-alone group (Figure 5A–B). Compared to the rPSOP25 group, there was no obvious difference in the cDC or pDC in the MPLA and QS-21 groups, and MPLA+QS-21 (Figure 5A–B). Using flow cytometry, we evaluated the percentage of DCs (CD11c⁺) in the spleen. As shown in Figure 5C, antigens formulated with MPLA, QS-21, and the two combination regimes didn’t influence the percentage of DCs. The Alum and CpG significantly expand the proportion of DCs compared with the rPSOP25 group (Figure 5C). The percentage of DCs in the CpG group was higher than the combination group QS-21+CpG. An increased level of mature DCs (CD11c⁺MHCII⁺ cells) was found in both the Alum and CpG groups (Figure 5D). The CpG adjuvant increased the level of MHC-II compared with the combination adjuvant QS-21+CpG and Alum adjuvant. These results support that the CpG adjuvant potentially triggers the activation and differentiation of DCs, suggesting that it may enhance the antigen-presenting activity of DCs.

Figure 5. Gating for DC uptake rPSOP25 vaccines in Splenocyte.

(A). The differentiation of cDC among the treatment rPSOP25 of various formulations. Mice were primed on day 0 and boosted on days 14 and 28 with or without various adjuvants of rPSOP25. Mice (n=3) were sacrificed 10 days after the third immunization, and splenocytes were isolated. The x-y axis refers to BV605-CD11c FITC-CD11b; cells were gated for CD11c⁺CD11b⁺. (B). The differentiation of pDC among the treatment rPSOP25 of various formulations. The x-y axis refers to BV605-CD11c PE-cy7-B220; cells were gated for CD11c⁺B220⁺. (C). The activation of DC by different vaccines. The x-y axis refers to Bv605-CD11c; cells were gated for CD11c⁺. (D). The activation of MHC-II⁺ in DC. The x-y axis refers to Bv605-CD11c APC-MHCII. cells were gated for MHC-II⁺CD11c⁺. Data were representative of three independent experiments.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test).

Th1 cells, activated by APCs, are crucial for activating phagocytes to eliminate resident pathogens [28]. CpG and QS-21 significantly promoted the percentage of Th1 (CD4⁺IFN-γ⁺) cells compared with rPSOP25 alone (Figure 6). The lack of a significant difference between single and combination adjuvant groups may be attributed to the halving of the adjuvant doses in the latter. As expected, the Alum group showed weaker activation of Th1 cells than other adjuvanted vaccines (Figure 6). These data implied that CpG may promote antigen uptake and facilitate interaction between DCs and T cells, thereby inducing a Th1 and Th2 immune response.

Figure 6. Gating for Th1 cells following immunization with various adjuvanted vaccines and rPSOP25 alone.

The proportion of Th1 cells and the dot plot shows the APC-IL-4 BV650-IFN-γ (x-y axis). The cells were gated for CD4⁺cells, and CD4⁺ were then gated for IFN-γ⁺ Representative plots are shown from experiments with n=3 mice. Data were representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test).

Tfh play a crucial role in the development of protective immunity and are critical for B cell activation, antibody class switching, and GC formation [29]. We assessed the proportions of Tfh and GC B cells in the spleens of mice from different vaccination groups. Flow cytometry assays demonstrated a significant increase in the ratio of Tfh cells (CD4⁺CXCR5⁺PD-1⁺) in the MPLA, CpG, and QS-21+CpG and Alum groups compared to the rPSOP25 group, while other vaccination groups did not have an impact on the proportions of Tfh cells (Supplementary Figure S3A). All adjuvant groups were capable of significantly enhancing the proportion of GC B cells (B220⁺CD95⁺GL-7⁺) in mice (Supplementary Figure S3B), with prominence seen in the Alum and QS-21+CpG groups. The QS-21+CpG group exhibited an increase of GC B cells compared to the single CpG or QS-21 groups, indicating a potential synergistic effect between these two adjuvants in promoting GC B cell formation.

We also evaluate the proportions of plasma cells in the spleens of mice from different vaccination groups. The adjuvant groups significantly promoted the percentage of plasma cells (B220⁺CD138⁺) compared with the rPSOP25 group. The QS-21+CpG group notably promoted the percentage of plasma cells compared with the QS-21, CpG, and Alum adjuvants, which aligns with the analysis of the IgG response. There was no difference between the other adjuvant groups and the Alum group (Figure 7).

Figure 7. Gating for splenic plasma cells following immunization with various adjuvanted vaccines and rPSOP25 alone.

The dot plot shows the PEcy7-B220 BV785-CD138 (x-y axis). cells were gated for B220⁺CD138⁺. Data were representative of three independent experiments. Representative plots are shown from experiments with n=3 mice. * P<0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (One-way ANOVA, Tukey post hoc test).

Memory cells are crucial components for vaccination activity. The proportion of central memory T cells was significantly elevated in the CpG and QS-21+CpG groups compared with the antigen-only group (P < 0.05). The proportion of central memory T cells was higher in the CpG group than in the Alum group. It appears that the CpG adjuvant can stimulate central memory T cells as effector T cells and enhance reactive memory in the presence of antigen (Supplementary Figure 4).

3.7. Evaluation of the long-term potency of elicited antibody

The durability of the antibody response is essential for malaria TBVs. Antibody levels were determined and compared in all vaccination groups at days 112 and 180 after the first vaccination. The antibody titers of all adjuvant groups were 1:256,000 at 112 days after the initial vaccination, whereas the rPSOP25 alone group exhibited an antibody titer of 1:128,000 (Figure 8A). At day 180, the adjuvant groups showed a sustained antibody response, as evidenced by the antibody titer reaching 1:256,000 versus 1:32,000 for the antigen-only group (Figure 8B). Adjuvant groups showed a sustained antibody response, while rPSOP25 showed a gradual decline in antibody levels at days 112 and 180 when compared with day 38 (Figure 8C). These results indicate that the adjuvanted vaccines performed better than antigen alone to induce a sustained antibody response.

Figure 8. The long-term potency of antibody response and antibody subclass in vaccinated mouse sera was assayed by ELISA.

BALB/c mice (n=3) were immunized subcutaneously with recombinant PSOP25 alone or formulated with different adjuvants (MPLA, CpG, QS-21, combination). Specific IgG titers (A-B) were analyzed on days 112 and 180 after primary immunization. (C) Antibody levels on days 112 and 180 compared with day 38. The IgG1 levels (D) and IgG2a levels (E) were detected in mouse serum at days 38, 112, and 180. The dashed line (cut-off value) indicates the mean of PBS antiserum +3×SEM. * P<0.05, ** P<0.01, *** P<0.001, (Two-way ANOVA, Tukey post hoc test).

To illustrate the potential of adjuvant-induced persistent antibody subclass responses, IgG1 and IgG2a levels were determined by ELISA at days 112, and 180 after the first vaccination. The data showed no significant difference in IgG1 levels between day 180 and day 38, even in the rPSOP25 group (Figure 8D). The IgG2a levels in the rPSOP25, MPLA, and Alum groups significantly decreased on days 112 and 180 when compared with day 38. However, other adjuvant groups exhibited sustained IgG2a levels, indicating a long-lasting antibody response (Figure 8E).

4. Discussion

Despite extensive research on the biology and immunology of malaria, the development of an efficacious vaccine for its control and eradication remains a formidable challenge. The efficacy of a recombinant vaccine depends on both an appropriate antigen and an optimal adjuvant to mediate the induction of long-term protective immune responses. In recent decades, researchers have focused on improving existing subunit malaria vaccines and developing new, effective formulations to enhance their immunogenicity and significantly reduce malaria transmission [30]. Adjuvants have been extensively employed in vaccines to enhance the efficacy of immunization and are considered essential in vaccine development, albeit with the mechanisms of action remaining incompletely elucidated [7]. Ample studies have demonstrated that Alum, MPLA, CpG ODN 1826, and QS-21 could work as effective adjuvants in cancer or infectious disease vaccines [11, 31]. Alum has been widely used in malaria vaccine development studies, although it lacks high adjuvanticity, and it is usually used as a reference for evaluating adjuvants in animal studies. Alum is a depot-type adjuvant, which functions by creating a reservoir and steady release of antigen. In malaria vaccine development, MPLA, CpG, and QS-21 or their combination adjuvant systems have been used successfully in several candidate vaccines [32, 33]. In this comparative study, the immunogenicity and functional efficacy of vaccines were evaluated with antigens formulated with various types of adjuvants in BALB/c mice. The results demonstrated their ability to promote high-titer antibodies that inhibit ookinete development and oocyst formation.

Recombinant protein, when used as an immunogen, typically induces only modest levels of antibodies and a weak cellular immune response due to the loss of its intrinsic immunological triggers during purification. The addition of adjuvants can impact the immunogenicity of antigens with respect to antibody responses, such as isotypes, titers, and avidity [34]. The ELISA results in our study demonstrated that the incorporation of adjuvants in the antigen formulation resulted in a modest enhancement of both antibody levels and avidity. The third immunization demonstrated better performance on antibody responses compared to the second, indicating that a single boost is insufficient for rPSOP25. It was shown that the low antibody avidity may be caused by inadequate stimulation of Toll-like receptors [35]. As TLR agonists, MPLA and CpG operate predominantly on TLR-4 and TLR-9, respectively. This study consistently showed that serum avidity significantly improved by vaccination with rPSOP25 with adjuvants, compared with antigen alone. The long-term efficacy of vaccines relies on the involvement of Tfh and GC B cells, which play a crucial role in maintaining the durability of the antibody response. The flow cytometry analysis revealed that the formulation with CpG or QS-21 led to a significant increase in GC B cells in immunized mice. MPLA potentially promotes the development of both Tfh and GC cells, which is in line with earlier findings [36]. These facts may contribute to the excellent sustainability of antibody response in the MPLA, CpG, and QS-21 groups, as evidenced by antibody titer remaining at 1:256,000 on day 180.

The quality and quantity of elicited antibodies have been reported to correlate with antibody functional activity [32]. The in vitro ookinetes culture studies demonstrated that antibodies induced by vaccination with the CpG or QS-21+CpG adjuvants exhibit relatively higher efficacy in inhibiting ookinete formation. The antiserum collected from the CpG group dramatically decreased the production of ookinetes and oocysts, and the CpG group exhibited the highest efficacy in TBA. These data demonstrated that CpG improves antigen immunogenicity and the protection effect, indicating it may be an effective adjuvant in malaria vaccine development.

Different adjuvants have been shown to induce and modulate different types of immune responses by inducing antigen-presenting capacity, promoting cytokine production, and enhancing humoral or cellular immune commitment. It has been demonstrated that CpG ODN promotes the innate immune response by activating the TLR-9 receptor on DCs, facilitates pDC maturation, and enhances antigen presentation, thereby inducing a robust immunological response and establishing immune memory in vaccinated mice. Cytokine analyses in the present study showed that CpG induced the production of IL-6, TNF-α, and IFN-γ, consistent with previous studies [37–39]. These proinflammatory cytokines may be linked to the generation of protective antibodies. The activation and differentiation of DCs found in the CpG group suggest it may enhance efficient antigen processing and presentation.

The advantage of Alum is its strong stimulation of antibody secretion. However, it has been associated with drawbacks, such as the inability to induce appropriate Th1-type immune responses, which are critical for eliminating pathogens. In contrast, CpG and MPLA specifically activate TLRs, producing proinflammatory cytokines and ultimately leading to a strong Th1-type cellular response [40]. QS-21 has been shown to induce both humoral and cellular immune responses by activating the NLRP3 inflammasome in APCs [19]. The evidence suggests that cell mediated immunity is effective in reducing malaria transmission. Cytokines associated with cellular immunity, such as IFN-γ, can help with antibody production and antibody switching that can help in inducing the cytophilic antibodies [27, 41]. A Th1 immune response appears to be important in producing inhibitory antibodies for oocyst formation, as demonstrated by the generation of a Th1 response with an increased expression of IFN-γ and TNF-α and high levels of IgG2a. The IgG1/IgG2a ratio approaches 2.0 in all of the adjuvant vaccine groups, and no statistically significant difference was observed when compared with the rPSOP25 antigen alone, indicated that the target adjuvants skew the immune responses toward a balanced Th1/Th2 response. CpG and QS-21 elevate the Th1 proportion in mice, along with enhancing their capacity to facilitate the maturation of dendritic cells. These may result in the ability of the mice to produce a better cellular immune response than Alum adjuvant, as evidenced by enhanced levels of proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α. The presence of IL-6 may promote the differentiation of Tfh cells, thereby effectively supporting the proliferation of B cells into plasma cells [42]. Further study is needed to characterize the time course of IL-21 induction after vaccination and the corresponding T-cell differentiation.

The history of the RTS,S vaccine has demonstrated that malaria vaccines formulated with a single adjuvant may not consistently elicit the requisite responses necessary for lasting and effective immunity. The utilization of combination adjuvants, such as liposome-based AS01, Matrix-M, and GLA-SE, has demonstrated enhanced antigen presentation and immune stimulation in malaria subunit vaccines [43]. Considering this, we combined the TLR agonists MPLA or CpG with QS-21. Unexpectedly, the combination groups did not significantly enhance immunogenicity compared to the single adjuvant groups. The only noteworthy finding is that the QS-21+CpG groups demonstrated a significant increase in GC B cells compared to the single CpG and QS-21 groups. The absence of evident synergistic effects among different types of adjuvants observed in our study with the two combination adjuvant groups may be attributed to several contributing factors. Firstly, comparing the AS01 or AS02 adjuvant systems, which consist of MPLA and QS-21 as their main components, demonstrated their ability to induce robust protective immune responses. AS01 or AS02 are formulated as liposomal or oil-in-water emulsions, which can protect antigens from degradation and prolong their bioavailability, resulting in APCs being able to capture more antigen signals. In this study, we merely combined these two individual adjuvants, which may have contributed to their weak protective immune responses. Another reason is that administering each adjuvant in the combination groups at a half dose might have resulted in comparatively weaker stimulation of immune responses.

Adjuvants could decrease the quantity of vaccine antigen and the times of immunization. In future studies, it will be important to optimize the adjuvant system to elicit a higher titer of high-affinity antibodies, which are capable of inducing strong TRA at a lower concentration. A better understanding of the properties and mechanisms of the action of adjuvants will facilitate the selection of an adjuvant capable of maximizing vaccine efficacy. This will expedite the development of vaccines for malaria and other infectious diseases. It is worth mentioning that not all antigens may be compatible with the same adjuvant. The determination of the “perfect” adjuvant is challenging, as each antigen may need to be tested with various types of adjuvants. Therefore, a rapid and judicious testing pipeline might be developed. Identification of the immunological correlates of vaccine efficacy can inform rational vaccine design and potentially accelerate clinical development. In summary, the results of this study reveal that CpG-ODN functions as a malaria vaccine adjuvant to boost DC maturation and effectively promote the level of GC B cells. It also strengthens the immune response to the antigen and prolongs the duration of antibodies, consequently enhancing TBA.

5. Conclusion

Our study provides a basis for understanding the action mechanisms of different types of adjuvants when formulation with malaria vaccine antigen PSOP25. PSOP25 subunit vaccines formulated with MPLA, CpG ODN 1826, QS-21, MPLA+QS-21, QS-21+CpG and Alum were systematically investigated and compared in terms of antibody production and protection efficacy in vaccinated mice. This study implies that CpG ODN 1826 may confer enhanced efficacy for the rPSOP25 protein vaccine, as evidenced by the elicited antiserum’s ability to induce protective immune responses and improved transmission-blocking activity.

Highlights.

• The immunogenicity and long-term immune response of rPSOP25 could be enhanced by conjugating with extrinsic adjuvants.

• CpG ODN 1826 adjuvanted vaccine demonstrated significant inhibition of ookinete formation and exhibited potent transmission-reducing activity.

• CpG ODN 1826 may confer enhanced efficacy for the rPSOP25 vaccine probably due to its capacity for enhancing activation and differentiation of dendritic cells.