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. 2025 Oct 21;16(1):2569621. doi: 10.1080/21505594.2025.2569621

Immunoregulation mediated by the enzyme inhibitory activity of helminth-derived serine protease inhibitor affects the protective efficiency of vaccines

Wenjie Shi a,#, Yan Liu b,#, Xue Bai a, Yue Liang a, Shuyan Zhang a, Lulu Sun a, Wei Lin a, Xiaolei Liu a, Ning Xu a,✉
PMCID: PMC12542607  PMID: 41117767

ABSTRACT

Recently, the prevalence of trichinellosis has increased the threat to animal food safety and public health worldwide. Pathogen-derived proteins are potential vaccine candidates for preventing and controlling Trichinella spiralis (T. spiralis) infection. However, the effect of protein-induced immunoregulation on vaccine protection remains unclear. In this study, a secretory protein derived from T. spiralis (Ts-serpin), whose enzyme inhibitory activity interfered with the host enzymatic reaction, was selected to reveal the way in which enzyme inhibitory activity regulates the immune system and its impact on the protective efficiency of vaccines. By modifying Ts-serpin, mutant proteins not only reduce the enzyme inhibition activity of Ts-serpin but also enhance the protective effect of vaccines in female BALB/c mice. The preference of mutant proteins for the inhibition on chymotrypsin and elastase affects different types of immune cells. In summary, the diverse inhibitory effects of Ts-serpin on serine proteases were shown to reduce the protective effects of active immunity by regulating the immune system. Our research provides an important basis for modifying protease inhibitors as vaccines to prevent infectious diseases and provides evidence for pathogenic invasion by interfering with host protease-related activity.

KEYWORDS: Trichinella spiralis, enzyme inhibitory activity, vaccine, immune protection, serine protease inhibitors

Introduction

Trichinellosis, an important zoonotic parasitic disease, is caused by the consumption of meat products containing infectious larvae [1]. In recent years, many countries have reported outbreaks of trichinellosis, which not only harms the global animal husbandry industry and reduces food security but also poses a serious threat to human health [2–4]. Two studies showed the high prevalences of Trichinella spiralis infection in domestic pigs, the main source of human infection, with 5.75% (Santa María Huazalotitlán, Mexico), 4.82% (San Andrés, Mexico) [5], and 0.9% (Central-Southern Chile) [6], respectively. Therefore, it is necessary to develop an effective vaccine to control trichinellosis. The development of T. spiralis from parent to offspring is completed in the same host. Its lifecycle is divided into the intestinal stage, circulatory system migration stage and muscle parasitic stage. The parasitism of T. spiralis larvae in the intestine is a prerequisite for their growth and development. The proteins secreted by worms during this period are important sources for screening vaccine candidate antigens [7,8]. According to the principle of species evolution, the proteins secreted by worms should assist in parasitism, which has been proven by many studies [9,10]. Foreign proteins have both regulatory and antigenic property on the host immune system. The immunogenicity of proteins ensures the establishment of immune protection, but the effect of immunoregulation mediated by protein activity on the protective efficiency of the vaccine is unknown. Hence, distinguishing the immune regulatory effects mediated by these two properties is necessary.

Serine protease inhibitor (serpin) secreted by worms can block the host enzymes to regulate the host response and protect themselves [11–13]. Serpin is a suicidal protease inhibitor characterized by irreversible enzyme inhibition. The reactive center loop (RCL) in the outer ring of the serpin protein structure contains key amino acids (P1 and P1”) that can be cleaved by proteases, and the different amino acid residues at P1 and P1” determine the specificity of protease inhibition [14]. Amino acids located before the amino-terminal of the P1 position were defined respectively as P2-P15. When the RCL substrate sequence is cleave by protease, it can interact with the proteases via covalent bonds. The RCL is then embedded inside the β-fold, in which the protease is pulled underneath the serpin to form a stable protein complex. During this process, proteases lose the oxyanion hole that maintains the stabilization of the tetrahedral intermediates, which directly leads to the loss of protease activity [15,16]. The properties of amino acid residues at P14-P12 can affect the rate of RCL insertion, which affects the inhibitory activity of the serpin [15]. This property provides a direction for studying the immunoregulatory mechanism of serpin inhibitory activity by modifying proteins.

A previous study identified a serpin derived from T. spiralis (Ts-serpin) with strong antigenicity from cDNA libraries of adults, newborn larvae (NBL) and muscle larvae (ML) [17], suggesting that Ts-serpin plays an important role in the whole parasitism process. Ts-serpin can induce alternative activation of macrophages, and this immunoregulatory effect contributes to the successful establishment of early infection [18]. In this study, we investigated the immunoregulatory effects mediated by the enzyme inhibitory activity of Ts-serpin. The key amino acid site of the Ts-serpin was predicted and mutated. Vaccine candidates were obtained to explore the role of immunomodulation mediated by enzyme inhibitory activity in the establishment of vaccine protection. The elucidation of the protein activity-dependence molecular mechanism not only preliminarily describes the immunoregulatory pathway mediated by enzyme inhibition activity, but also provides new insights for improving the immune protection of serpin vaccines.

Materials and methods

Bioinformatics analysis and preparation of recombinant proteins

Amino acid multiple alignment of serpin was performed by Jalview software (Table S1). The sequences of the two Ts-serpin mutants (Ts-serpin-d1 and Ts-serpin-d2) were obtained by point mutation of the key amino acids. The spatial structures of the Ts-serpin protein (GenBank: DQ864973.2) and mutant proteins were predicted by SWISS-MODEL [19–23]. Recombinant proteins, with the exception of the signal peptide, were inserted into the expression vector pET-28a. The constructed vectors were subsequently transferred into BL21(DE3) cells, which were then stimulated with isopropyl-β-d-thiogalactoside (Solarbio LIFE SCIENCES) to express protein. The soluble recombinant protein was obtained from bacterial lysate by NI-column affinity chromatography (Cytiva). The purified recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue, and no other protein bands were found (Figure S1).

Detection of enzyme inhibitory activity

A total of 20 μg of Ts-serpin/ Ts-serpin-d1/ Ts-serpin-d2 was incubated with an equal amount of elastase/chymotrypsin (Sigma-Aldrich) for 10 minutes, and 200 nmol of chromogenic substrate (Sigma-Aldrich) was added to a total volume of 200 μL for the detection of enzyme inhibitory activity. After the chromogenic substrate was added, the absorbance of the mixture was immediately analyzed with an ELISA plate reader (BioTek). The protease reacted with the corresponding substrate as the enzyme activity control, and the substrate alone was detected as the negative control. The chromogenic substrates of elastase were analyzed at a wavelength of 405 nm. The chromogenic substrate of chymotrypsin was analyzed at a wavelength of 410 nm. Based on the maximum absorbance value of the enzyme activity curve of chymotrypsin or elastase with the substrate, the reaction time at half of the maximum absorbance value was selected to analyze the difference in the absorbance of the different treatment groups.

Animals and parasites

The protein-immunized experiment was conducted with three independent times, and there were five mice in each independent experiment group. Results from one representative independent experiment (five mice) were shown, and results from other independent experiments were provided in the supplementary data (animal experiment start – end time: 7 August 2023 - 23 October 2023). BALB/c mice aged six- to eight-week-old were procured from the Jilin University Experimental Animal Center (Jilin, China). OT-II ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic C57BL/6 mice were procured from Model Animal Research Centre of Nanjing University (Nanjing, China). All the animal experiments were conducted in compliance with the guidelines for the care and use of laboratory animals [24]. All the animals were given adequate food and water, and were kept in the same environment. The animal experiments were approved by the Ethical Committee of Jilin Medical University which is affiliated with the Provincial Animal Health Committee (Jilin, China; ethical clearance no. 2023-GKJJ001). The T. spiralis (ISS534) from our laboratory was used in the experiment.

Immunization protocol for the recombinant proteins

The immune program of the mice was performed in accordance with previous studies [25]. Five BALB/c mice were randomly kept in same cage according to the group. Briefly, the combination of 50 μg/50 μL recombinant proteins (Ts-serpin/ Ts-serpin-d1/ Ts-serpin-d2) and 50 μL Freund adjuvant (Sigma-Aldrich) immunized mice by subcutaneous injection on Day 0, 14, and 28. PBS or adjuvant immunized mice were used as natural infection controls to evaluate the immune protective effect, and untreated mice were used as blank control. A week after the protein immunization program ended, the mice were infected with 250 T. spiralis except for blank control mice. Serum samples were collected on Day −7, 7, 21, 35, and 70 for antibody titer and cytokine detection. Spleen and peritoneal cavity cells on Day 70 were collected for flow cytometry. Muscle larvae were collected from the mice on Day 70 and the steps were as follows. Mouse fur and viscera were removed, and all muscle tissue was retained. The muscles were cut and digested with a solution containing 1% pepsin and 1% hydrochloric acid for 3 hours at 37 °C. ML were then obtained by the washed precipitation method, and the number of ML in the 20 μL of solution was counted under a microscope and finally converted to the number of larvae in the total solution volume. Six days after ML infection, Ad6 adults in the intestine were collected as described below. The small intestine of mice was collected and placed in 0.9% saline at 37 °C for 3 hours. The small intestine was subsequently removed and adult worms at the bottom of the solution were counted.

Detection of cytokine levels and specific antibody

The protein- specific IgG, IgG1, and IgG2a levels in the serum were measured by indirect ELISA (experiment time: 9 January 2024). In brief, 100 μL of recombinant protein (5 μg/mL) was added to the 96-well plates and incubated overnight at 4 °C. The plates were blocked with 5% skim milk for 2 hours at 37 °C. The diluted serum or cell culture supernatant were added and incubated for 1 hour at 37 °C. HRP-conjugated anti-mouse IgG, IgG1, and IgG2a (Abcam) were diluted and incubated for 1 hour at 37 °C. Then, 100 μL of TMB substrate was added and reacted for 10 minutes, after which 50 μL of stop solution was added. The absorbance of the 96 wells was analyzed at 450 nm. The positive serum was determined according to the ratio of the diluted serum OD value to the negative serum OD value being greater than or equal to 2.1 [26,27].

The cytokines IFN-γ, IL-4, IL-5, IL-13, IL-10, IL-17A, TNF-α, and IL-2 were detected with Luminex (R&D Systems), and the mouse TGF-beta1 indirect ELISA kit (Proteintech) was used to detect the level of TGF-β1 (experiment time: 5 March 2024).

Histology of the diaphragmatic muscle

The diaphragmatic muscle was collected and fixed with 4% paraformaldehyde for 24 hours. Sections of partial diaphragmatic muscle was stained by hematoxylin and eosin (H&E). Muscle larvae cysts were observed with a microscope. Inflammatory cell infiltration in cysts was scored according to the scoring system from absent (0), minimal (1), moderate (3), to marked (4). The rest of the diaphragmatic muscle was placed onto slides and then examined with a microscope. The number of cysts in the random field of view was counted, and the number of cysts per square millimeter of diaphragm was calculated.

Isolation and culture of mouse primary cells

The leg bones and spleens of euthanized OT-II mice were collected to isolate primary cells. First, bone marrow cells were isolated in advance and induced to differentiate into bone marrow-derived dendritic cells (BMDCs) in vitro. Red blood cells were removed from the bone marrow cells by red blood cell lysis (Solarbio LIFE SCIENCES), and the bone marrow cells were cultured in RPMI-1640 medium (Procell system) supplemented with 10% fetal bovine serum (Procell system), 25 ng/mL rmGM-CSF (PeproTech®), and 10 ng/mL rmIL-4 (PeproTech®). Three-quarters of the culture medium was replaced with the same fresh culture medium on Day 2, 4, and 6. The OVA323–339 (Sigma-Aldrich, 25 μg) and recombinant protein (25 μg) were added to the medium on Day 6, after which the mixture was incubated for 24 hours to collect BMDCs. Naïve CD4+ T cells and B cells were then isolated from the spleen to construct a coculture system with the collected BMDCs. EasySepTM mouse Naïve CD4+ T-cell isolation kits (STEMCELL) and EasySep™ mouse B-cell isolation kits (STEMCELL) were used to isolate the naïve CD4+ T cells and B cells from single splenocyte suspensions according to the manufacturers’ instructions. BMDCs, naïve T cells, and B cells were added to the culture plate at a ratio of 2:2:1, and incubated for 48 hours. The cell colonies were observed and counted using an inverted microscope, and B-cell activation was then detected by flow cytometry.

Flow cytometry

Spleen and peritoneal cavity cells were collected from the mice on Day 70. Erythrocytes were removed by red blood cell lysates (Solarbio LIFE SCIENCES), and single-cell suspensions were obtained through a 70 μm filter. Peritoneal cavity cells were incubated with Fc receptor blocking antibodies (BD Biosciences) for 10 min. The cell surface molecules were stained with antibodies for 30 minutes. Next, the cells were fixed with 4% paraformaldehyde for 30 minutes and treated with 0.1% Triton X-100 for 40 minutes. The cells were stained with antibody for 30 minutes to detect intracellular molecules. The cells were washed with PBS three times and detected with a BD FACSCalibur flow cytometer. The antibodies are shown in Table S2.

Statistical analysis

GraphPad Prism 5 software was used to analyze the data and the results were presented as the means ± standard deviations (SDs). Normality and lognormality tests were used to analyze normality of data. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to analyze the differences in data between adjuvant-immunized group and other groups, and significant differences were regarded as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; ns: no significant difference. Unpaired t test was performed to analyze the differences in data between Ts-serpin-immunized group and mutant protein-immunized groups, and significant differences were regarded as follows: # p < 0.05; ## p < 0.01; ### p < 0.001; ns: no significant difference.

Results

Design and functional activity of mutant proteins

Multiple sequence alignment of amino acids revealed that the Ts-serpin amino acid numbers (P15-P1) and the amino acid residues (P15-P9) were conserved (Figure 1(A)). The RCL region of Ts-serpin was predicted to be the amino acids 321 ~ 351, in which Met335 (P1) and Ser336 (P1’) were the cleavage sites of serine protease, and Thr322 (P14) and Ala324 (P12) were the key sites responsible for the enzyme inhibitory activity of the protein. Therefore, Ts-serpin-d1 and Ts-serpin-d2 were obtained by mutating Thr322 and Ala324 to Glu respectively (Figure 1(B)). Protein spatial structure prediction revealed that the mutated sites were located at the corner of the β-sheet, and amino acid mutation did not affect the protein’s spatial structure (Figure 1(C)). Ts-serpin strongly inhibits chymotrypsin and elastase, and the ability of the mutant proteins to inhibit elastase and chymotrypsin was weakened (Figure 1(D,E)). Interestingly, Ts-serpin-d1 showed stronger deactivation on elastase, whereas Ts-serpin-d2 display a preference for inactivating the chymotrypsin (Figure 1(D,E)).

Figure 1.

Figure 1.

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Bioinformatics analysis and functional activity of proteins. (A) A partial illustration of amino acid multiple sequence alignment of Ts-serpin with mammalian serpins. Red frame: the amino acids at the P15-P1 sites. (B) Mutation strategies for key amino acid sites in Ts-serpin. (C) The predicted spatial structures of Ts-serpin, Ts-serpin-d1, and Ts-serpin-d2. Dashed lines area: the spatial position of the mutated amino acid in the protein structure. The enzyme inhibitory effects of the recombinant proteins on elastase (D) And chymotrypsin (E). The left panel shows the absorbance detection for time continuity, and the right panel shows the statistical plot of absorbance at a single time point.

The immunization of mice with mutant proteins can reduce the load of muscle larvae

After stimulating active immunity with the original or mutated recombinant proteins, the mice were artificially infected with T. spiralis (Figure 2(A)). The parasite load of ML is as the gold standard for evaluating the immune protection of proteins. Direct microscopic examination and H&E staining of the diaphragmatic muscle sections were used to observe and evaluate the infection status of ML. T. spiralis infection successfully achieved parasitism by larvae in muscle tissue, and the use of the adjuvant had no significant influence on larval survival (Figure 2(B)). The observation of diaphragmatic muscle revealed that three protein-immunized groups, including the Ts-serpin-immunized, Ts-serpin-d1-immunized and Ts-serpin-d2-immunized groups, presented significantly low parasite loads, especially Ts-serpin-d1-immunized and Ts-serpin-d2-immunized groups (Figure 2(B)). Compared with the adjuvant control group, the muscle tissue of the protein-immunized groups showed more inflammatory cell infiltration. Furthermore, a mass of inflammatory cells could be observed to have entered the cysts and contacted the ML (Figure 2(C)). The number of intestinal Ad6 adults and ML in mouse muscle clearly confirmed that amino acid mutations caused changes in the protective effect of Ts-serpin as a vaccine (Figure 2(D)). The average reduction rate of ML was 55.565% in the Ts-serpin-immunized mice, 71.480% in the Ts-serpin-d1-immunized mice, and 67.270% in the Ts-serpin-d2-immunized mice (Figure 2(E)).

Figure 2.

Figure 2.

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Parasite load of protein-immunized mice. (A) Time points of protein immune protection experiments. (B) Light microscopy images of the diaphragmatic muscle and the statistical plot of the number of cysts per square millimeter. The dashed lines area indicates the cyst of the ML. (C) Images of H&E sections of diaphragmatic muscle and the statistical plot of inflammatory score of cysts. Dashed lines area: cyst of the ML. (D) The total amount of worms in the intestine on Day 6 and ML in muscle tissue on Day 35 after infection. (E) The proportion of reduced ML load in mice of each experimental group compared with PBS + T. spiralis group. The data came from five mice.

Mutant proteins affect the levels of specific antibodies and cytokines in the serum

The immune efficiency of the three proteins was evaluated by the antibody levels. Unlike the absence of an antibody response to adjuvant immunity alone (Table S3), the specific-IgG antibody titers after three immunizations were higher than 128,000 in three protein-immunized groups (Figure 3(A)). The three proteins induced similar specific-IgG levels, but the specific-IgG1 and specific-IgG2a levels exhibited different changes after the immunization program (Figure 3(B,C)). Briefly, Ts-serpin-d1 and Ts-serpin-d2 induced higher specific-IgG1. Ts-serpin-d1 induced lower specific-IgG2a while Ts-serpin-d2 induced similar level of IgG2a than Ts-serpin (Figure 3(C)).

Figure 3.

Figure 3.

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The levels of specific antibodies and cytokines in the serum. The detection of specific-IgG (A), IgG1 (B), and IgG2a (C) Levels in the serum after immunizing mice with proteins. The red line represents 2.1 times the average OD value of the negative control serum. (D) The statistical analysis of cytokine levels in the serum. The data came from five mice.

The cytokine levels of IFN-γ, IL-4, IL-5, IL-13, IL-10, IL-17A, TNF-α, IL-2, and TGF-β1 were determined on Day 70, which provides references for evaluating the immune status. The levels of IFN-γ, IL-5, IL-17A, TNF-α, and IL-10 in the Ts-serpin-immunized group were greater than those in the adjuvant-immunized group, whereas the levels of IL-2 and TGF-β1 were lower than those in the adjuvant-immunized group (Figure 3(D)). Interestingly, the levels of IL-13, IL-17A, TNF-α, and TGF-β1 in the Ts-serpin-d1-immunized group were significantly lower than those in the Ts-serpin-immunized group, whereas the level of IL-2 was greater than that in the Ts-serpin-immunized group (Figure 3(D)). Additionally, the levels of IL-5, IL-10, and IL-17A in the Ts-serpin-d2-immunized group were lower than those in the Ts-serpin-immunized group, whereas the levels of IFN-γ and IL-2 were greater than those observed in the Ts-serpin-immunized group (Figure 3(D)).

Enzyme inhibition activity of Ts-serpin induces the differentiation of peritoneal macrophages

Peritoneal macrophages are the key innate immune cells that regulate the host immune response. The use of the adjuvant alone decreased the percentage of F4/80+ macrophages, and this change in the three protein-immunized groups was more significant (Figure 4(A,B)). In accordance with the fluorescence intensity of F4/80, macrophages were divided into two subsets: F4/80hi macrophages and F4/80int macrophages [28–31] (Figure S2A). The reduced proportion of F4/80hi macrophages was the main cause of the reduced proportion of F4/80+ macrophages (Figure 4(A,C)). In addition, the Ts-serpin-d2-immunized group had a significantly lower proportion of F4/80hi macrophages than the Ts-serpin-immunized group did (Figure 4(C)). In contrast, the use of adjuvant increased the proportion of F4/80int macrophages, and the addition of proteins further promoted this trend, especially in the Ts-serpin-d2-immunized group (Figure 4(D)). Next, the macrophage subsets among the F4/80int macrophages were analyzed. The three protein-immunized groups presented a high proportion of F4/80int CD16/32+ CD206+ macrophages, and the Ts-serpin-d2-immunized group presented a weaker increasing trend than the Ts-serpin-immunized group did (Figure 4(E,F)).

Figure 4.

Figure 4.

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The differentiation of macrophage subsets in the peritoneal cavity. (A) The cell points plot of two F4/80+ macrophage subsets (F4/80hi macrophages and F4/80int macrophages). The cell proportion statistics of F4/80+ macrophages (B), F4/80hi macrophages (C), and F4/80int macrophages (D) are shown. (E) The cell points plot of F4/80int CD16/32+ CD206+ macrophages. (F) The percentages of F4/80int CD16/32+ CD206+ macrophages are shown. The data came from five mice.

Mutant proteins have different abilities to affect the immune cells in the spleen

The differentiation of immune cell subsets in the spleen was detected to elucidate changes in the host immune status (Figure S2B, C). The use of proteins and Freund adjuvant had no effect on the proportion of CD45+ immune cells in the spleen (Figure 5(A)). The proportion of B220+ CD4− B cells was significantly reduced in the three protein-immunized groups and the fluorescence intensity of B220 on the cell surface decreased significantly in the adjuvant-immunized and three protein-immunized groups (Figure 5(B)). T. spiralis infection significantly reduced the proportion of CD3+ T cells, and Ts-serpin-immunization exacerbated this decreasing trend (Figure 5(C)). However, although the Ts-serpin-d1-immunized and Ts-serpin-d2-immunized groups restored the reduced proportion of CD3+ T cells and increased the ratio of CD4+ T cells to CD8+ T cells compared with the Ts-serpin-immunized group, the regulatory ability of Ts-serpin-d1 was significantly greater than that of Ts-serpin-d2 (Figure 5(C, D)). The proportion of CD4+ Foxp3+ regulatory T (Treg) cells significantly increased in the groups infected with T. spiralis, and there was a greater proportion of Treg cells in the Ts-serpin-immunized group than in the Ts-serpin-d1/ Ts-serpin-d2-immunized groups (Figure 5(E)).

Figure 5.

Figure 5.

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Changes in immune cell subsets in the spleen. (A) The cell points plot and cell proportion statistic of CD45+ immune cells. (B) The cell points plot and cell proportion statistic of B220+ CD4− B cells. (C) The cell points plot and cell proportion statistic of CD3+ T cells. (D) The cell points plot and the statistical ratio of CD4+ T cells to CD8+ T cells. (E) The cell points plot and cell proportion statistic of CD4+ Foxp3+ Treg cells. The data came from five mice.

Enzyme inhibition activity of Ts-serpin inhibits the activation of B cells in vitro

BMDCs, purified CD4+ naïve T cells and B cells were cocultured to study the regulatory effect of the enzyme inhibitory activity of Ts-serpin on the adaptive immune response (Figure 6(A)). OVA323–339 can stimulate cells to form colonies, which is the basis by which BMDCs activate CD4+ naïve T and B cells (Figure 6(B)). Ts-serpin had a strong ability to inhibit the formation of cell colonies, and Ts-serpin-d1 restored part of the cell colony, which was related to its enzyme-inhibiting activity (Figure 6(B)). Interestingly, Ts-serpin-d1 and Ts-serpin-d2 had different regulatory effects on the formation of cell colonies, which may be due to differences in their ability to inhibit different proteases. Consistent with the above results, Ts-serpin-d1 can restored the high expression of CD19 and the production of IgG antibody in B cells, but Ts-serpin and Ts-serpin-d2 strongly inhibited CD19 and IgG (Figure 6(C,D); and Figure S2D). Therefore, the enzyme inhibitory activity of Ts-serpin affects the activation of B cells, which may be the cause of the differences in the levels of antibody subtypes in the serum of protein-immunized mice.

Figure 6.

Figure 6.

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The enzyme inhibitory activity of the Ts-serpin affects the activation of B cells in vitro. (A) Diagram of the cell coculture experiment. (B) Cell images and cell colony statistics after 24 hours of culture. (C) Representative plots and percentages of CD19hi B cells. (D) OVA323–339-specific IgG levels in cell culture supernatants.

Discussion

Enzymatic reactions are the important methods of information transmission in animals and are rapid, efficient, and extensive. These types of systems play important roles in the complement system [32], clotting system [33], and immune system [34,35]. The secretion of serpin by worms to regulate the host enzyme reaction is an important mean of parasitism [36]. Hence, blocking this pathway is one of the important directions for the development of recombinant protein vaccines. However, most studies of recombinant protein vaccines have neglected the regulatory effect of the enzyme inhibitory activity of serpin on the establishment of immune protection [37,38]. In this study, we weakened the ability of Ts-serpin to inhibit the hydrolytic activity of proteases via protein mutation. The acquisition of two mutant proteins with different inhibitory capacities for chymotrypsin and elastase has laid a foundation for studying the immunomodulatory effects mediated by the inhibitory activities of different proteases. We then studied the regulatory effect of protease inhibitory activity on the establishment of immune protection by active immunization. The results showed that attenuating the protease inhibitory activity of Ts-serpin can increase vaccine effectiveness by affecting innate and adaptive immune responses.

According to multiple sequence alignment, the amino acids at the P1/P1’ site of the Ts-serpin are Met and Ser, which are the cleavage sites of elastase and chymotrypsin [15], as confirmed by the results of enzyme inhibition activity in this study. The amino acids at the P14 site or P12 site were mutated to the charged Glu to reduce the rate at which RCL was inserted into the β-fold, thereby reducing the enzyme inhibitory activity of the protein [15]. Unexpectedly, Ts-serpin-d1 and Ts-serpin-d2 had different abilities to inhibit the enzyme activity of elastase and chymotrypsin. The amino acid mutation at individual sites did not affect the spatial structure of the protein, which suggests that they can act as immunogenic inducers of host protection. Our results confirmed that the preservation of the immunogen properties of Ts-serpin-d1 and Ts-serpin-d2 maintained their ability to induce immune protection. The parasite load and state of ML are the gold standards for evaluating the protection efficacy of vaccines [39]. The recombinant protein-immunized groups present high reduction rate of ML, a large number of inflammatory cells infiltrated around the cyst, and the greater protective effect of immunization with Ts-serpin-d1 and Ts-serpin-d2 suggested that the enzyme inhibitory activity reduced the protective effect of the vaccine.

The protective efficacy of vaccines depended on the activation of host innate and adaptive immune responses by immunized proteins. High levels of antibodies, which are representative effector molecules against pathogens, are key to long-term immune protection, and the levels of different antibodies reflect changes in the immune state [40,41]. In this study, consistent with the increase in specific- antibodies, the proportion of B220hi B cells (B2 cells) in the spleen that produced specific antibodies against heterologous stimuli [42–45], was significantly decreased in three protein-immunized groups, which suggested that B2 cells might be recruited into the bloodstream to exert a humoral immune response. Evaluation of different antibody levels revealed that Ts-serpin-d1 and Ts-serpin-d2, with reduced enzyme inhibition, had the ability to induce higher levels of specific-IgG1 and lower levels of specific- IgG2a than Ts-serpin. These results suggested that the enzyme inhibitory activity of Ts-serpin was involved in immunoregulation, which caused a class-switch between IgG1 and IgG2a.

Since T. spiralis infection begins with invasion of the gut, peritoneal macrophages play an important role in early resistance against worms and influence subsequent adaptive immune responses [18,46]. It has been reported that elastase contributes to promoting the early inflammatory cascade response and inflammatory cell infiltration [47]. Briefly, the degradation of vascular endothelial growth factor-A into different fragments by elastase enhances the chemotaxis of macrophages and monocytes to inflammatory tissue [48,49]. These studies imply that the ability of Ts-serpin-d2 but not Ts-serpin-d1 to induce more monocyte-derived F4/80int macrophages may be related to elastase. In addition, elastase can affect the phagocytic function of macrophages and promote the inflammatory response by degrading histone deacetylases and sirtuins [50,51]. In our study, the weakened inhibitory capacity of Ts-serpin-d2 on elastase reduce the proportion of anti-inflammatory F4/80hi large peritoneal macrophages and F4/80int CD16/32+ CD206+ macrophages. These results implied a regulatory pathway in which the inhibitory effect of Ts-serpin on elastase can regulate macrophages, thereby mediating a weak immune response to resist T. spiralis.

Adaptive immunity is an effective mean for the host to specifically remove the pathogens. The results of coculture in vitro directly proved that the enzyme inhibitory activity of Ts-serpin inhibited the activation of B cells by preventing the formation of immune cell colonies, which explained the differences in antibody responses to immunization with different mutant proteins. Ts-serpin-d1 can induce B cell activation in vitro, but the total IgG antibody level of immunized mice was similar to that of Ts-serpin-immunized mice, suggesting that the antibodies produced by plasma cells of Ts-serpin-d1-immunized mice were more extensive in their recognition of the antigen epitope spectrum, which is related to the establishment of better immune protection efficiency. T cells are the key executors of the cellular immune response, and the differentiation of T cells affects the immune status of the host [52]. Loss of chymotrypsin activity inhibits the ability of DCs to take up antigens and damaged costimulatory factors (CD80 and CD86) on the membrane surface [53]. Combined with the mutant protein’s bias for chymotrypsin and elastase, it is possible that chymotrypsin is important for immune cell aggregation. Chymotrypsin is also necessary for T cells to produce IL-2, which is a key cytokine that promotes the proliferation of T cells [54]. Similar to the results of in vitro cell experiments, mutant proteins induced a high proportion of total CD3+ T cells and CD4+ T cells and a low proportion of Treg cells, accompanied by reduced TGF-β1 and IL-10 levels in the serum. Interestingly, the changes of the two anti-inflammatory cytokines (IL-10 and TGF-β1) in the two mutant protein-immunized groups were different, which might be caused by the changes of different immune cell subsets, such as peritoneal F4/80hi macrophage and Treg cells in the spleen. In addition, high levels of IFN-γ and IgG2a in the serum of Ts-serpin-d2-immunized mice suggested a strong type I immune response. The elastase activity of neutrophils contributes to the differentiation of Th1/Th17 cells and promotes the transformation of Treg cells into Th1/Th17 cells [55], which is consistent with the strong type I immune response in the Ts-serpin-d2-immunized group, but there was no significant change in IL-17A level. In summary, Ts-serpin can regulate the adaptive immune system by inhibiting the activities of elastase and chymotrypsin.

In conclusion, this study proposed two mutation schemes in which the enzyme inhibitory activity of Ts-serpin was reduced with no changes in spatial conformation, which enhanced immune protection by inducing a strong immune response. The detection of antibody levels and immune cell types suggested that two mutant proteins differentially regulated the immune system, which was related to differences in the reduced enzyme inhibitory activity by the mutation. These differences in immunoregulation caused by mutant proteins suggest that the enzyme inhibitory activity of Ts-serpin contributes to regulate the immune system in multiple ways, providing a direction for vaccine modification of other parasites or pathogen-derived serpin.

Supplementary Material

Figure S1.tif
KVIR_A_2569621_SM6264.tif (179.7KB, tif)
Supporting Information.docx
KVIR_A_2569621_SM6263.docx (19.4KB, docx)
Table S2.docx
KVIR_A_2569621_SM6262.docx (18.4KB, docx)
Supplementary data.xlsx
KVIR_A_2569621_SM6261.xlsx (32.6KB, xlsx)
Table S1.docx
KVIR_A_2569621_SM6260.docx (16.3KB, docx)
Figure S2.tif
KVIR_A_2569621_SM6259.tif (2.7MB, tif)
Table S3.docx
KVIR_A_2569621_SM6258.docx (17.1KB, docx)

Funding Statement

This work was supported by the National Key Research and Development Program of China [grant number 2023YFD1802400, 2023YFC2308603]; the National Nature Science Foundation of China [grant number NSFC 82302563, 32202835, 32373032]; State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases [grant number 2024ZZ00010]; State Key Laboratory for Animal Disease Control and Prevention (grant number SKLADCPKFKT202514).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Abbreviations

Trichinella spiralis

T. spiralis;

Excretory/secretory products,

ESPs;

muscle larvae,

ML;

newborn larvae,

NBL;

reactive center loop,

RCL;

regulatory T cells,

Treg cells;

T. spiralis-derived serine protease inhibitors,

Ts-serpin.

Data availability statement

The data that support the findings of this study are openly available in [figshare] at https://doi.org/10.6084/m9.figshare.29673416.v1 [56].

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2569621.

ARRIVE guidelines

The manuscript has adhered to ARRIVE guidelines.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.tif
KVIR_A_2569621_SM6264.tif (179.7KB, tif)
Supporting Information.docx
KVIR_A_2569621_SM6263.docx (19.4KB, docx)
Table S2.docx
KVIR_A_2569621_SM6262.docx (18.4KB, docx)
Supplementary data.xlsx
KVIR_A_2569621_SM6261.xlsx (32.6KB, xlsx)
Table S1.docx
KVIR_A_2569621_SM6260.docx (16.3KB, docx)
Figure S2.tif
KVIR_A_2569621_SM6259.tif (2.7MB, tif)
Table S3.docx
KVIR_A_2569621_SM6258.docx (17.1KB, docx)

Data Availability Statement

The data that support the findings of this study are openly available in [figshare] at https://doi.org/10.6084/m9.figshare.29673416.v1 [56].

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