EgLDH as a Novel Target: Design and Preliminary Efficacy Assessment of a DNA Vaccine

TL;DR

This study designs a DNA vaccine targeting lactate dehydrogenase in Echinococcus parasites and shows it reduces liver cysts in mice, offering a potential new approach for controlling echinococcosis.

Contribution

The novel contribution is the design and preliminary efficacy assessment of a DNA vaccine targeting EgLDH, a key enzyme in the parasite's energy metabolism.

Findings

The EgLDH DNA vaccine reduced liver cysts in mice by 80.95% compared to the control group.

The vaccine elicited increased IgG and specific cytokine responses (IL-1, IL-4, IL-10) in immunized mice.

Abstract

Echinococcosis has been classified by the World Health Organization (WHO) as a priority zoonotic parasitic disease to be eradicated before 2050. This study focuses on a central aspect of the parasite’s energy metabolism—the Embden–Meyerhof–Parnas pathway (EMP)—selecting a key enzyme, lactate dehydrogenase (LDH), as a target for the development of a DNA vaccine and evaluating its immunogenicity.

(1) Background: Echinococcosis is a significant zoonotic disease that the World Health Organization (WHO) aims to eliminate by 2050. Current drug-based control faces challenges such as drug resistance, highlighting the urgent need to develop vaccines as a supplementary strategy. Although some progress has been made in the study of intermediate host vaccines using antigens such as Eg95, there is still no commercial vaccine available for the definitive host, canines—which are crucial for transmission—and it is not yet suitable for large-scale use. While vaccine studies targeting the key enzyme lactate dehydrogenase (LDH) in parasite energy metabolism remain scarce, they represent a promising area of potential. (2) Methods: The B cell antigen epitopes of LDH were analyzed, and prokaryotic (pET-28a-EgLDH) and eukaryotic (pVAX1-EgLDH) DNA vaccine expression vectors were constructed. After verifying expression and immunogenicity via qRT-PCR and WB, in vitro validation was performed in 293T cells. Mice were immunized with the vaccine and then challenged with the parasite; blood was collected from the orbital sinus, and IgG levels and cytokines were measured by ELISA. Protective effects were assessed through counting liver cysts and histopathological analysis. (3) Results: We constructed the pVAX1-EgLDH plasmid and immunized Kunming (KM) mice. Compared with the PBS control group, the vaccine group showed an 80.95% reduction in liver cysts (Quil-A group: 19.00%). Histopathological analysis indicated no significant liver damage, although the spleens in the vaccine group were smaller. ELISA results demonstrated an increase in total IgG (p < 0.05), and cytokine analysis showed elevated levels of IL-1 (p < 0.01), IL-4, and IL-10 (p < 0.001), whereas IL-5 and IFN-γ showed no significant changes (p > 0.05). (4) Conclusions: The EgLDH DNA vaccine can elicit a specific immune response and significantly reduce cyst burden, providing theoretical basis and data support for its use as a candidate vaccine for the prevention and control of Echinococcosis.

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1. Background

Echinococcosis, recognized as a globally significant zoonotic disease, has been identified by the World Health Organization (WHO) as a priority for eradication by 2050 [1]. Cystic Echinococcosis (CE), caused by the cestode Echinococcus granulosus (E. granulosus), poses a substantial challenge to public health and economic stability owing to its widespread geographic distribution and diverse range of intermediate hosts [2,3]. Human infection occurs through accidental ingestion of eggs, resulting in cyst formation within visceral organs such as the liver, while canids act as definitive hosts, playing a crucial role in the transmission cycle [4,5]. Current control strategies predominantly involve the administration of anthelmintic treatments to canines (e.g., praziquantel) and the enforcement of hygiene regulations in slaughterhouses. Although these methods have demonstrated short-term efficacy, they face emerging challenges, including the potential development of anthelmintic resistance and the risk of long-term pharmacological residue accumulation [6,7]. This underscores the urgent need for the development of sustainable complementary interventions. Vaccination is an effective complementary strategy to control the spread of Echinococcosis [8].

The development of a vaccine targeting the intermediate host aims to inhibit cyst formation in livestock, thereby mitigating the risk of intermediate host infection through the consumption of contaminated grass or water [9]. Current research has focused on several key antigens including EgDf1 [10], Eg95 [11], EgG1Y162 [12], and Eg14-3-3 [13]. These antigens can elicit an immune response in intermediate hosts, thereby enhancing their resistance to parasitic infections. Meanwhile, ongoing studies have indicated the potential of candidate vaccine antigens, such as EgHCDH [14], the EgM protein family [15], EgA31 [16], EgTrp [17], and EgTSP11 [18], for definitive hosts. However, further studies are needed to assess the long-term efficacy and safety of these vaccines. A whole-genome analysis of E. granulosus [19] revealed that its energy metabolism is heavily reliant on the Embden–Meyerhof–Parnas pathway (EMP). Lactate dehydrogenase (LDH), a core enzyme linking the EMP, may represent a “metabolic vulnerability” that could be targeted to disrupt the survival of the parasite [20,21]. Research using gene knockout technology to knock out the LDH gene indicates that LDH has a decisive influence on the growth and development of Toxoplasma and Microsporidia, further confirming its importance in the physiological functions of parasitic organisms in the body [22,23]. LDH is crucial to promoting the development of new vaccines for Echinococcosis. Current research efforts are predominantly concentrated on Plasmodium, Toxoplasma gondii, and so on, whereas studies focusing on E. granulosus remain relatively limited [24]. DNA vaccines have the advantages of strong targeting, high safety, and a simplified production process [25], and they are expected to circumvent the limitations of traditional vaccines, serving as a new strategy for the prevention and control of CE. One key study was conducted by Yan and colleagues, who cloned, expressed, and enzymatically characterized the LDH isoenzymes of E. granulosus and Echinococcus multilocularis (EgLDH-A/B, EmLDH-A/B). This study revealed differences in the structure and kinetics of these isoenzymes and provided an initial assessment of their potential as drug targets [26]. In this study, E. granulosus lactate dehydrogenase (EgLDH) was used as the target and the DNA vaccine pVAX1-EgLDH was constructed. The vaccine, in combination with the Quil-A (QA) adjuvant, enhanced the immune response. The Kunming (KM) mice were immunized to obtain preliminary immune effects. Given the role of EgLDH in parasite metabolism, the selection of EgLDH as a candidate vaccine represents a promising and rational strategy, which serves as a reference for subsequent development.

2. Methods

2.1. Detection of EgLDH Transcriptional Levels

The relative transcription levels of the EgLDH gene at different developmental stages of E. granulosus were detected using qRT-PCR. The primers of the EgLDH gene and GAPDH reference gene are shown in Table S1. The reaction system (20 μL) consisted of 2xTaq Pro Universal SYBR qPCR Master Mix, qEgLDH-F/qGAPDH-F 1 μL, qEgLDH-R/qGAPDH-R 1 μL, cDNA 2 μL, and ddH_2_O 6 μL. Reaction procedure: preincubation at 95 °C for 3 min, two-step amplification for 10 s; 60 °C for 30 s; melting at 95 °C for 10 s; cooling to 60 °C for 1 min; and cooling to 37 °C for 30 s. This procedure was repeated for a total of 45 cycles.

2.2. Cloning and Expression of EgLDH

PCR primers were designed based on the EgLDH gene sequence provided in the NCBI database (sequence accession number: HM748917). The underlined parts indicate the EcoR I and Hind III restriction sites and protective bases. Primers for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH, sequence accession number: XM_024494574).

We removed the adult E. granulosus and PSCs, which were frozen in TRIZOL reagent (Thermo Fisher, Waltham, MA, USA) in a laboratory freezer at −80 °C, and extracted the RNA using the Ultrapure Total RNA Extraction Kit (Cowin, Taizhou, China), according to the manufacturer’s instructions. The cDNA First-Strand Synthesis Kit (Tiangen, Beijing, China) was used for reverse transcription of the first strand of cDNA, according to the manufacturer’s instructions. PCR amplification was performed with the EgLDH primers. The PCR product was electrophoresed on a 1.5% agarose gel for 30 min at 120 V and 400 mA. The target band was recovered using the agarose gel DNA recovery kit (Tiangen, Beijing, China) and ligated with the pMD19-T vector (Takara, Kyoto, Japan) overnight at 4 °C. The ligation products were transformed into the cloned DH5α competent cell strain (Tiangen, Beijing, China) and incubated at 37 °C overnight for 12 h. Subsequently, single colonies were picked and cultured at 180 rpm for 4 h. After PCR identification, the culture was expanded, and the plasmid was extracted using a plasmid mini-extract kit (Tiangen, Beijing, China). The recombinant plasmid was named pMD19-T-EgLDH, as identified by EcoR I and Hind III double digestion and sequencing. The above steps were followed to construct pET-28a-EgLDH. The pET-28a-EgLDH plasmid was transformed into BL21 competent cells (Tiangen, Beijing, China), induced by IPTG (Sangon, Shanghai, China) for 8 h. The bacterial solution was subjected to cell lysis, the supernatant and precipitate were separated, the precipitate was dissolved in 8 M urea, and this solution was subjected to His-Ni affinity column chromatography (Solarbio, Beijing, China). The EgLDH recombinant protein was purified by column chromatography, reconstituted, and concentrated in dialysis bags. The molecular weight and expression of the protein were determined using a SDS-PAGE kit (Cowin, Taizhou, China). Western blot (WB) was performed to evaluate the immunogenicity of the purified recombinant EgLDH protein, using positive serum from E. granulosus-infected dogs (1:100) as the primary antibody and an HRP-conjugated rabbit anti-dog IgG (Solarbio, Beijing, China) at a 1:2000 dilution as the secondary antibody.

2.3. Construction of Eukaryotic Expression Plasmid pVAX1-EgLDH

The eukaryotic vector plasmid pVAX1 was transformed into DH5α competent cells, and the transformed bacterial solution was inoculated into 5 mL of LB liquid medium containing Kanamycin for 8 h of cultivation. A plasmid Mini Extraction Kit was used to extract plasmids. The recombinant plasmid, pMD19-T-EgLDH, was used for inoculation and cultivation, and extraction was performed after 8 h.

2.4. Plasmid Transfection of 293T Cells

The pVAX1-EgLDH DNA plasmid was transfected into 293T cells using Lipofectamine 2000. After 24 h and 48 h post-transfection, the 293T cells were collected and centrifuged to isolate the cell pellet, which was then placed in TRIZOL Reagent for RNA extraction and reverse transcription into cDNA. Transcription was examined using semi-quantitative PCR and qRT-PCR.

2.5. KM Mice Used for Parasite Infection

The KM mice used in this study were purchased from the Experimental Animal Center of Xinjiang Medical University. All mice were SPF-grade, female, and aged 6–8 weeks. The experiment was divided into three groups (5 mice per group): the PBS group (200 μL of PBS), QA group (50 μg of QA in 150 μL of PBS), and the pVAX1-EgLDH DNA vaccine group (50 μg of pVAX1-EgLDH in 50 μg of QA and 100 μL of PBS). Under the established experimental conditions, relevant research data show that the carrier has good safety in mice. Under the established experimental conditions, relevant research data show that the carrier has good safety in mice [27]. Immunization was performed through intramuscular injection in KM mice, with each injection being 200 μL, administered every 15 days (on days 0, 15, and 30). Blood was collected from the mouse orbit before each immunization, centrifuged at 3000 rpm for 5 min, and the serum was stored at −80 °C for future use. Thirty days after completion of the three immunizations, 2000 viable E. granulosus PSCs (washed with PBS) were intraperitoneally injected into the vaccinated mice. These PSCs were extracted from sheep hydatid cysts obtained from a local slaughterhouse, and their viability was confirmed using Trypan Blue staining. Ninety days post-infection (150 days after immunization), the KM mice were euthanized, and the number of liver hydatid cysts and spleen sizes was measured.

2.6. Detection of Antibodies and Cytokines

KM mice serum was retrieved from the −80 °C freezer and allowed to thaw naturally. If any precipitate appeared, the mixture was centrifuged to collect the supernatant. The sample was diluted to one-third of the original concentration (50 μL of serum mixed with 100 μL of sample diluent). ELISA was performed to measure the levels of IgG, IL-1, IL-4, IL-5, IL-10, and IFN-γ in mouse serum.

2.7. Check the Number of Liver Cysts

After 90 days post-infection (150 days after immunization), the KM mice were sacrificed, their livers removed, and the liver cysts counted. We calculate the cyst reduction rate according to (1).

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2.8. Statistical Analysis of Spleen Changes

Ninety days post-insect attack (i.e., 150 days post-immunization), KM mice were euthanized. Subsequently, their livers were excised, and the number of hepatic cysts was counted. The KM mice were sacrificed; the spleens removed, the spleen morphology was observed, and the spleen was measured and weighed.

3. Results

3.1. B Cell Antigen Epitope Prediction for EgLDH Protein

The results of the IEDB online software (Bepipred Linear Epitope Prediction 2.0) prediction indicated that the EgLDH protein had 13 potential B cell antigen epitopes located at amino acid positions 6–18, 56–58, 72–85, 98–108, 125–125, 150–153, 195–198, 204–228, 242–242, 278–283, 307–310, 312–313, and 324–324 (Figure 1). This suggests that the EgLDH protein has good antigenicity.

3.2. Cloning and Prokaryotic Expression of EgLDH

To carry out immunological research, we first successfully prepared high-purity recombinant EgLDH protein using a prokaryotic expression system.

A specific band consistent with the expected size appeared at 996 bp in the amplification of the EgLDH gene (Figure 2), which was consistent with the expected size. Recombinant plasmids pMD19-T-EgLDH (Figure 3a) and pET-28a-EgLDH (Figure 3b) were constructed and identified by double digestion with the restriction endonucleases EcoR I and Hind III, which indicated that the recombinant plasmids were successfully constructed.

SDS-PAGE was performed to detect the recombinant protein pET-28a-EgLDH. The results revealed a distinct band at 40 kDa (Figure 4). The recombinant EgLDH protein was successfully expressed. Western blotting revealed that the recombinant EgLDH protein showed an immunological reaction with the positive serum of dogs infected with E. granulosus; a positive band appeared at 40 kDa (Figure 5), proving that the recombinant protein exhibited good antigenicity.

3.3. Analysis of the Transcriptional Levels of EgLDH

To assess the transcriptional activity of EgLDH, we found that its expression during the larval stage was significantly higher than in the adult stage, suggesting that this gene may play a key role in the parasite’s hypoxic metabolism and energy supply.

Fluorescence-based qRT-PCR was performed to detect the transcriptional levels of the EgLDH gene at different developmental stages of E. granulosus (Figure 6). The transcriptional level of this gene was extremely significantly higher in the adult worm stage than in the protoscolex stage (p < 0.01).

3.4. Gene Expression in 293T Cells

To verify the expression ability of the constructed pVAX1-EgLDH DNA vaccine plasmid in eukaryotic cells, we transiently transfected the plasmid into 293T cells.

The eukaryotic vector pVAX1-EgLDH was transfected into 293T cells utilizing the transfection reagent Lipofectamine 2000. Verification using semi-quantitative PCR and qRT-PCR demonstrated successful transcription of the vector in 293T cells (Figure 7 and Supplementary Figure S5), with the highest transcription level observed 48 h post-transfection (p < 0.05) (Figure 8).

3.5. Antibody Responses in Immunized KM Mice

To evaluate the overall humoral immune response elicited by the DNA vaccine, we collected mouse serum at different time points after immunization and measured the total serum IgG levels and cytokine changes in the serum by ELISA.

Changes in the antibody levels in KM mice were detected using ELISA. At 15, 30, 60, and 150 days after immunization, the secretion of total IgG antibodies in the pVAX1-EgLDH DNA vaccine group was higher than in the QA and PBS groups. Compared with the PBS group, the pVAX1-EgLDH DNA vaccine group showed a significantly enhanced secretion of total IgG antibodies at 150 days after immunization (i.e., after challenge with parasites) (p < 0.05). Compared with the QA group, the pVAX1-EgLDH DNA vaccine group showed no significant change in total IgG antibodies at 15, 30, and 60 days after immunization (p > 0.05) but showed a significant increase in the secretion of total IgG antibodies at 150 days (p < 0.05). Total IgG in the pVAX1-EgLDH DNA vaccine group showed an upward trend at 15, 30, 60, and 150 days, reaching its highest level at 150 days compared with day 0 (p < 0.05; Figure 9), which indicates that it successfully elicited a strong humoral immune response. Total IgG significantly increased (p < 0.05) at 15, 30, 60, and 150 days but slightly decreased at 60 days. The overall trend of total IgG antibodies in the pVAX1-EgLDH DNA vaccine group was upward; however, total IgG showed a slight decrease at 60 days.

3.6. Cytokine Production in Immunized KM Mice

ELISA was performed to detect changes in the levels of the antibodies IL-1, IL-4, IL-5, IL-10, and IFN-γ in KM mice (Figure 10A–E). The pVAX1-EgLDH DNA vaccine group showed a significant enhancement in IL-1 secretion at 15 and 60 days (p < 0.01) and an extremely significant enhancement at 30 days (p < 0.001). The pVAX1-EgLDH DNA vaccine group showed a significant enhancement in IL-1 secretion at 15 and 60 days (p < 0.01) and an extremely significant enhancement at 30 (p < 0.001) and 150 days (p < 0.0001). The IL-1 secretion level in the vaccine group was lower than that in the QA group but higher than that in the PBS group. The pVAX1-EgLDH DNA vaccine group showed an extremely significant enhancement of IL-4 secretion at 15 (p < 0.001), 30 (p < 0.001), 60 (p < 0.001), and 150 days (p < 0.0001). IL-5 secretion was not significantly enhanced in the pVAX1-EgLDH DNA vaccine group (p > 0.05). The pVAX1-EgLDH DNA vaccine group showed a significant enhancement in IL-10 secretion (p < 0.001) at 30 days, 60 days, and 150 days. However, the enhancement of secretion was not significant after 15 days (p > 0.05). IFN-γ secretion was not significantly enhanced in the pVAX1-EgLDH DNA vaccine group (p < 0.05); however, compared with the vaccine group, the PBS group showed a significantly higher secretion at 60 days (p < 0.01) and an extremely significantly higher secretion at 150 days (p < 0.0001).

3.7. Cyst Burden in the Liver of KM Mice

To assess the protective efficacy of the vaccine, we counted liver cysts in infected mice and collected spleen tissues for histological analysis.

At 90 days post-parasitic challenge (150 days post-immunization), KM mice were euthanized for liver cyst enumeration. The vaccine group exhibited a significant reduction in hepatic cyst burden compared to the control groups (Table 1 and Figure 11). In terms of mean values, the number of liver cysts in the pVAX1-EgLDH DNA vaccine group was substantially lower than that in the QA and PBS groups. With the PBS group serving as the control, the cyst reduction rate in the pVAX1-EgLDH DNA vaccine group was 80.95%, whereas that in the QA group was 19.05% (p < 0.05).

3.8. Comparison of Spleen Size and Weight Between Groups of Mice

The spleens of the mice in each group were collected 150 days after post-infection to observe changes in their size and morphology (Table 2). The average length and weight of the spleens of the KM mice in the pVAX1-EgLDH DNA vaccine group were less than those in the PBS and QA groups; however, the difference was not significant (p > 0.05).

4. Discussion

To address the challenges associated with the low efficacy of current prevention and control measures, as well as the development of vaccines for hosts of E. granulosus, this study focuses on the development of vaccines targeting the disease [1,2,3] and intermediate hosts of the disease. This study specifically selected the key enzyme LDH, which is crucial for the EMP in the parasite [21]. Protective immunity against E. granulosus infection is primarily mediated by antibodies, and the identification and utilization of its specific B cell-protective epitope peptides is of great significance for vaccine development [28,29]. This study found that EgLDH has 13 potential antigenic epitopes. Western blotting results indicated that the recombinant EgLDH protein could be recognized by the serum of dogs infected with E. granulosus, suggesting that the EgLDH protein has good antigenicity. Additionally, studies have indicated that EgLDH is a potential target for vaccines against E. granulosus [20]. In the present study, we successfully cloned and expressed the EgLDH gene. The expression of this gene in the adult stage was significantly higher than that in the metacestode stage, indicating that the EMP process is more active in adult E. granulosus than in metacestodes.

In mouse models, DNA vaccines can elicit a strong immunogenic response [30]. Therefore, we developed an EgLDH DNA vaccine to induce an effective immune response. Using ELISA to detect antibody levels, we found that the total secretion of IgG antibodies in the pVAX1-EgLDH DNA vaccine group was significantly increased (p < 0.05), indicating that this vaccine could effectively initiate an antibody response in the KM mice. The genetic diversity of E. granulosus is rich at the species level. However, no effective drugs are available for CE [31]. Currently, research is primarily focused on several key antigens. EgDf1 [32] encodes a fatty acid-binding protein involved in lipid transport within the organism. It is responsible for the production of significantly high levels of Th-2 cytokines, suggesting its potential as a candidate vaccine for E. granulosus. The Eg95 [33] recombinant protein vaccine has been commercially produced and utilized in certain countries to achieve good control. Following immunization with the EgG1Y162 [34] peptide, the levels of IFN-γ and IL-4, as well as the IFN-γ/IL-4 ratio, significantly increased, enhancing the protective immunity of mice against E. granulosus infection. Therefore, the EgG1Y162 gene may serve as a potential candidate vaccine target. However, research on EgLDH is limited. One key study was conducted by Yan and colleagues, who cloned, expressed, and enzymatically characterized the LDH isoenzymes of E. granulosus and Echinococcus multilocularis (EgLDH-A/B, EmLDH-A/B). This study revealed differences in the structure and kinetics of these isoenzymes and provided an initial assessment of their potential as drug targets [26]. This offers important insights for functional studies of LDH. This study used molecular cloning, prokaryotic expression, and Western blotting. By evaluating changes in antibody levels, cyst reduction rates, and Th1/Th2 cytokine levels related to the immunoprotective effects of EgLDH in mice, we determined the antigenicity of EgLDH.

Cytokines are the core of the immune regulatory network [35], and in CE, host–parasite interactions are strictly regulated by the dynamic balance of Th1/Th2 responses [36]. This study measured the inflammatory factor IL-1, the Th1 factor IFN-γ, and the Th2 factors IL-4, IL-5, and IL-10. The results showed that the pVAX1-EgLDH DNA vaccine significantly increased total IgG levels in mice (p < 0.05) and specifically enhanced the secretion of IL-1, IL-4, and IL-10 (p < 0.01), while IL-5 and IFN-γ showed no significant changes. CD4^+^ T cells play a central regulatory role in parasitic infections through cytokines. IL-1 supports sustained CD4^+^ T cell responses, while IL-4 is crucial for resistance against CE cysts [37]. In this study, IL-4 and IL-10 were observed to increase simultaneously. It is noteworthy that, although IFN-γ in the vaccinated group did not increase significantly, it remained at a stable level of 25–35 pg/mL, indicating that the immune response exhibited a mixed state based on IFN-γ activity, accompanied by Th2 regulatory cytokines, rather than a purely Th2-dominant response. This sustained Th1 activity may support anti-parasitic effects, while the Th2 shift in the later stages of infection might facilitate immune evasion by E. granulosus [38].

Compared with the PBS control group, the pVAX1-EgLDH vaccine achieved a cyst reduction rate of 80.95%, significantly higher than the QA group (19.00%) and the EgA31 [38] vaccine (50–60%). Although slightly lower than the classic surface antigen target Eg95 at around 98% [39], its mechanism of interfering with energy metabolism by targeting a key glycolytic enzyme is a notable feature. This endogenous target can act on multiple developmental stages of the parasite, complementing the surface-blocking mechanism of Eg95 [40] and providing a new idea for combined immunization strategies.

In addition, the spleen size and weight of mice in the EgLDH DNA vaccine group were slightly lower than those in the PBS and QA groups, but the differences were not significant (p > 0.05), which may be related to the shorter infection period. This vaccine primarily exerts its protective effect by inhibiting the early colonization and development of parasites. Therefore, its core effect is reflected in a significant reduction in parasite burden, while the excessive inflammatory response induced by the infection is also alleviated once the parasites are controlled. Given the limited sample size and short observation period of this study, it is difficult to systematically assess chronic adverse reactions, and further in-depth research is needed.

5. Conclusions

The EgLDH vaccine stimulated antibody production, induced humoral and cellular immunity, and effectively reduced the burden of liver cysts in mice. However, owing to the short duration of infection, no significant differences were observed in the pathological changes in the liver sections and the average spleen length and weight. Therefore, future research on relevant vaccines requires the further development of immunization schedules. The results of this study provide a basic reference for future research on E. granulosus vaccines.