Immunogenicity Evaluation of Six Key Antigens of Mycoplasma pneumoniae in BALB/c Mice

TL;DR

This study evaluates six Mycoplasma pneumoniae proteins in mice to identify potential vaccine candidates based on their ability to induce immune responses and reduce lung damage.

Contribution

The study identifies CARDS, P116, and MPN133 as top vaccine candidates due to their strong immunogenicity and protective effects against MP infection.

Findings

Six MP proteins induced strong antibody responses in mice.

P116 and MPN133 showed the strongest MP growth inhibitory activity.

CARDS, P116, and MPN133 significantly reduced lung injury in mice.

Abstract

Background/Objective: Mycoplasma pneumoniae (MP) remains a significant pathogen causing respiratory tract inflammation, particularly in the elderly and children under 5 years old. The lack of an effective vaccine is primarily attributed to the absence of well-defined immunological targets. This study systematically evaluated the immunological characteristics of six key MP proteins to facilitate vaccine development. Methods: We constructed recombinant plasmids (pET30a-CARDS/P1/P40-90/P30/P116/MPN133), expressed them in Escherichia coli, and evaluated their immunogenicity and immune reactivity against MP infection in BALB/c mice. Results: Six proteins induced strong antibody responses. Sera from all protein-immunized groups exhibited MP growth inhibitory activity, with P116 and MPN133 showing more pronounced inhibitory effects. After MP challenge, lung histopathological findings demonstrated that, except for the P1 group, the lung pathological scores of the protein-immunized groups were lower than those of the PBS control group, and the lung injury in the CARDS, P116 and MPN133 groups was significantly alleviated. Conclusions: The six recombinant proteins demonstrate good immunogenicity. Among them, CARDS induces a strong primary antibody response, while P116 and MPN133 elicit potent anti-MP antibodies. In addition, CARDS, P116 and MPN133 significantly alleviate lung pathological damage (n = 6, p < 0.01), indicating that these three proteins have greater potential as MP vaccine targets among the six candidate proteins.

Figures

Full text

1. Introduction

Mycoplasma pneumoniae (MP) is a naturally cell wall-deficient prokaryotic microorganism that can cause respiratory inflammation and extra-pulmonary complications in humans [1,2]. Elderly individuals, children under 5 years old and immunocompromised populations are the most susceptible groups [3,4]. Mycoplasma pneumoniae pneumonia (MPP) accounts for 20% to 30% of all community-acquired pneumonia (CAP) cases [5,6,7]. Macrolide antibiotics are the first-line treatment for MP infections. However, since 2000, the global incidence of macrolide-resistant MP infections has increased rapidly, particularly in Asian countries, with resistance rates in China reaching as high as 69% to 95% [8,9,10,11]. The treatment-resistant pneumonia caused by MP in children poses a serious threat to their health [12,13,14]. Moreover, multiple studies have shown that MP exhibits periodic epidemics, with peaks occurring every 3 to 5 years, placing a significant burden on clinical healthcare systems [15,16]. To date, no MP vaccine has been marketed. The main challenges facing MP vaccine development include concerns over its efficacy, stability and safety [17]. Research has shown that inactivated MP vaccines carry a risk of vaccine-enhanced diseases post-immunization [18]. In response to these challenges, identifying more suitable vaccine targets may represent an effective solution.

MP mediates cellular adhesion primarily through surface adhesion proteins and specialized adhesion organelles. This process compromises the integrity of respiratory epithelium, leading to reduced ciliary motility, increased vacuolation and shedding of cells [19,20]. Key adhesion proteins of MP include P1, P30, P116 and other auxiliary adhesion proteins, such as P40, P90, as well as high-molecular-weight proteins HMW1 and HMW3 [21]. Both the P1 and P30 proteins exhibit similar structural functions and play critical roles in MP’s adhesion and motility. The P116 protein serves as another major MP antigen and a crucial cell adhesion factor. Importantly, anti-P116 antibodies demonstrate the capacity to independently inhibit MP from attachment to Hep-2 cells, irrespective of P1 [3]. Due to their strong immunogenicity, P1, P30, and P116 are commonly used for MP serological diagnosis and vaccine development. Furthermore, the auxiliary adhesion proteins P40-90 also show strong reactivity with sera from infected humans. The structural characteristics of P1 and P40-90 present novel opportunities for developing vaccines against MP infection [19]. MP also produces the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, an exotoxin possessing ADP-ribosyltransferase and vacuolating activities, which contributes significantly to MP pathogenesis. Several studies have reported that the C-terminal region of the CARDS toxin can induce specific antibody production, with higher antibody titers during the convalescent phase [22,23]. Additionally, MPN133, a nucleic acid enzyme with cytotoxic activity, plays a unique role in MP lifecycle-related processes and in inducing host cell pathological damage [24,25].

Therefore, this study aims to express the above six proteins via the Escherichia coli expression system and evaluate their immunological characteristics through animal experiments, thereby facilitating the development of MP vaccines.

2. Materials and Methods

2.1. Target Genes, Standard Strains, and Competent Cells

The sequences of CARDS (accession no.: 4TLW_A), P1 (WP_010874498.1), P40-90 (WP_010874499.1), P30 (WP_010874809.1), P116 (GAB1890787.1) and MPN133 (WP_010874490.1) were downloaded from the NCBI database. The classic experimental strain of MP (M129) is preserved by the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). The Escherichia coli BL21 (DE3) prokaryotic expression strain was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

2.2. Animals and Animal Study

Forty-two SPF-grade BALB/c mice (female, 6–8 weeks old, weighing 18–20 g) were purchased from the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). The animal qualification certificate and animal ethics approval numbers are SYXK(DY)K2022–0002 and DWLL202406014, respectively.

The mice were randomly divided into 7 groups, with 6 mice in each group. Mice in groups 1 to 6 were intramuscularly injected with 100 μL protein-adjuvant mixture, containing 10 μg of purified protein and 50 μg of aluminum hydroxide adjuvant. The control group received 100 µL of PBS via the same injection route. Immunization consisted of two doses administered 21 days apart. Blood samples were collected from the retro-orbital plexus 21 days after the prime/boost immunization. The serum was separated and stored at −20 °C. At 21 days after the boost immunization, mice were anesthetized with isoflurane and then challenged with MP via intranasal instillation (1 × 10^7^ CCU/mL, 100 µL per mouse).

2.3. Construction of Recombinant Plasmids, Protein Expression and Purification

The CARDS, P1, P40-90, P30, P116, and MPN133 genes, along with the pET30a vector were digested by NdeI and BamHI. Following digestion, the target gene fragments and vector fragments were recovered and ligated overnight at 16 °C using T4 DNA ligase. The ligation products were transformed into DH5α competent cells, which were cultured overnight at 37 °C. Single colonies were picked and sent to Sangon Biotechnology Co., Ltd. (Shanghai, China). for sequencing. Recombinant plasmids with correct sequencing (pET30a-CARDS/P1/P40-90/P30/P116/MPN133) were transformed into BL21 (DE3) competent cells, followed by overnight culture at 37 °C. Single colonies were selected for small-scale culture and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mmol/L to induce protein expression at 37 °C for 4–5 h. Target protein bands were verified by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting (WB) (WB using Abs specific to a 6 × His tag moiety). The positive clones were stored as glycerol stocks at −80 °C.

For large-scale protein preparation, the glycerol stocks were inoculated into 5 mL of LB medium containing kanamycin (50 mg/mL) for overnight activation culture. Subsequently, the culture was diluted 1:100 into 400 mL of fresh LB medium containing kanamycin. Cells were grown at 37 °C with shaking until the optical density at 600 nm (OD_600_) of the bacterial culture reached 0.6, IPTG was added with final concentration of 1 mmol/L, and the protein expression was performed overnight at 20 °C with shaking at 160 rpm/min. The induced bacterial culture was centrifuged at 6000 rpm for 10 min at 4 °C to collect the bacterial pellets, which were resuspended in 60 mL of PBS and then disrupted using a high-pressure homogenizer under the conditions of a lateral pressure of 100 MPa and a forward pressure of 550–650 MPa; the disrupted mixture was subsequently subjected to centrifugation at 7000 rpm for 1 h at 4 °C. After confirming that the target protein was present in the supernatant via SDS-PAGE, the supernatant was loaded onto a nickel ion affinity chromatography column equilibrated with PBS buffer (pH 7.4) containing 20 mmol/L imidazole at a loading flow rate of 1 mL/min. Gradient elution was performed using an imidazole concentration gradient ranging from 20 to 500 mmol/L, and the fractions corresponding to the OD_280_ absorbance peak were collected. High-purity fractions were identified by SDS-PAGE, then transferred to an ultrafiltration centrifugal tube, centrifugally concentrated at 4000 rpm and 4 °C, and subjected to buffer exchange with PBS.

If the target protein is present in the precipitate, it indicates that the protein exists as insoluble aggregates, namely inclusion bodies. In this case, it is necessary to resuspend the inclusion bodies in a denaturation buffer (8 M urea in PBS buffer, pH 8.5) and incubate the mixture overnight at 4 °C to ensure complete solubilization of inclusion bodies. Subsequently, gradient dialysis is performed: the protein solution is subjected to dialysis at 4 °C with a series of refolding buffers containing sequentially decreasing concentrations of urea (6 M, 4 M, 2 M, 1 M and 0 M in PBS buffer, pH 7.4), each dialysis step lasting no less than 4 h. After dialysis, the solution is centrifuged at 12,000 rpm for 20 min; the precipitate is discarded, and the resulting supernatant is the refolded soluble protein. The subsequent purification procedures for this refolded protein are identical to those used for the soluble protein from the aforementioned supernatant.

2.4. Antigen Specific Antibody Detection

Recombinant proteins CARDS, P1, P40-90, P30, P116 and MPN133 were diluted to 2 µg/mL in coating buffer (1.6 g/L Na_2_CO_3_; 2.9 g/L NaHCO_3_). Subsequently, 100 µL of each protein solution was added per well of a 96-well polypropylene (PP) microplate and incubated overnight at 4 °C. Then, 300 µL of 1% bovine serum albumin (BSA) was added to each well for blocking at 37 °C for 2 h. The plate was washed once with phosphate-buffered saline containing 0.05% Tween 20 (PBST), 300 µL per well. Mouse serum was diluted (starting at a 1:100 dilution in the first well, 1:1000 in the second and then serial two-fold dilution), 100 µL per well and incubated at 37 °C for 1 h. After incubation, the plate was washed 4 times with PBST, 300 µL per well. Then, HRP-conjugated goat anti-mouse IgG (1:10,000 dilution) was added (100 µL per well) and incubated at 37 °C for 1 h. The plate was washed 4 times with PBST, 300 µL per well. Next, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added, 100 µL per well and incubated at room temperature for 5–8 min. Finally, 50 µL 2 mol/L H_2_SO_4_ was added to stop the reaction and the OD_450_ was measured using an Enzyme-Linked Immunosorbent Assay (ELISA) reader (SpectraMax^®^ 190 Absorbance Microplate Reader, Sunnyvale, CA, USA).

2.5. MP-Specific Antibody Detection

The MP was centrifuged at 10,000 rpm for 50 min. The pellet was resuspended in PBS and washed 3 times. The pellet was resuspended in 10 mL of PBS and then subjected to ultrasonic disruption using an ultrasonic cell disruptor. The ultrasonic parameters were set as follows: power 350 W, working mode of 3 s sonication followed by 5 s interval, and total sonication duration 30 min. After disruption, the protein concentration of the product was measured, and it adjusted to 10 µg/mL in coating buffer. Aliquots (50 µL) of the diluted solution was added to a 96-well polypropylene microplate and incubated overnight at 4 °C. The blocking and washing procedures were performed identically to those described in Section 2.4. Mouse serum dilution was 1:100 in the first well, followed by serial two-fold dilution across subsequent wells. The secondary antibody incubation and color development were carried out as described in Section 2.4. The calculation method for the MP-specific binding antibody titer is as follows: the absorbance of serum from the PBS control group at a 1:100 dilution is used as the cut-off value, and the serum dilution corresponding to an absorbance 2.1 times this cut-off value is defined as the antibody titer.

2.6. Metabolic Inhibitory Antibody Detection

The procedure for detecting the activity of metabolism-inhibiting antibodies is as follows: Experimental preparation stage: Prepare MP medium (BD Difco™ Dehydrated Culture Medium, Cat. No. BD 255420; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 56.4 µM phenol red indicator (pH 7.9), collect serum from mice after booster immunization, and prepare a sterile 96-well cell culture plate. Serum treatment and addition: Dilute the serum to be tested in MP medium in a 2-fold series (8 gradients are set), add 50 µL of the diluted serum to each well in columns 1–7 of the 96-well plate, and add 50 µL of medium to each well in column 8 as MP growth control; simultaneously, dilute stationary phase MP 1:10 with medium and add 50 µL to all wells in columns 1–8. Standard curve setup: Using 1:10 diluted MP as the stock solution, prepare a MP with concentrations ranging from 90% to 0% (10 gradients in total), and add 100 µL to columns 9–10 of the 96-well plate. Incubation and detection: Place the 96-well plate in a 37 °C, 5% CO_2_ incubator, observe the color change in the medium in column 8 daily, and terminate culture when it turns yellow; measure the OD_560_ absorbance values of all wells using a microplate reader. Result determination: Fit a “MP concentration—OD_560_” standard curve using data from columns 9–10, compare the OD_560_ values between the experimental and control groups. OD_560_ values are negatively correlated with the activity of inhibitory antibodies in the serum; the titer of inhibitory antibodies is defined as the highest serum dilution that inhibits the metabolism of 50% of MP.

2.7. Pathological Examination of Lung Tissue

Following sacrifice, the lung tissues were immediately dissected and immersed in 4% paraformaldehyde fixative, for 24 h at 4 °C. After fixation, the tissues were dehydrated via a graded alcohol series (70% ethanol for 2 h, 85% ethanol for 1 h, 95% ethanol twice for 1 h each time, and anhydrous ethanol twice for 30 min each time), followed by clearing in xylene (twice for 30 min each time) in a fume hood with strict duration control to prevent tissue brittleness. Subsequently, the tissues were infiltrated with molten paraffin at 56–58 °C (twice, with the second infiltration lasting 1–2 h), embedded in embedding molds with proper tissue orientation, covered with freshly molten paraffin, and cooled at room temperature to form solid paraffin blocks. The paraffin-embedded blocks were cut into 4–5 μm thick sections using a microtome, and routine hematoxylin-eosin (H&E) staining was performed prior to observation. Lung tissue injury was scored based on four indicators: (1) necrosis, (2) inflammatory cell infiltration, (3) alveolar wall thickening, and (4) hemorrhage. Each indicator was scored from 0 to 4 according to the degree of pathological changes, with specific criteria as follows: 0 points = no abnormality or extremely mild abnormality; 1 point = mild and focal pathological changes; 2 points = moderate and focal pathological changes; 3 points = extensive moderate changes or focal significant pathological changes; and 4 points = extensive and significant pathological changes.

2.8. Statistical Analysis

Statistical analysis and data visualization were conducted using Microsoft Excel 2021, GraphPad Prism 8, and Adobe Illustrator 2024. The values are presented as the means ± standard errors of the means (SEMs). Differences among multiple groups were assessed using one-way analysis of variance (ANOVA) combined with Tukey’s post hoc test for pairwise comparisons. A p-value < 0.05 was considered statistically significant. Lung pathological scores were defined as semi-quantitative ordinal categorical data. After verification by the Shapiro–Wilk normality test and Levene’s test for homogeneity of variances, the data were confirmed to be non-normally distributed with heterogeneous variances. Therefore, nonparametric tests were employed for intergroup difference comparisons. The overall differences among multiple groups were analyzed using the Kruskal–Wallis H test. A value of p < 0.05 was considered statistically significant, and p < 0.01 was considered extremely statistically significant.

3. Results

3.1. Recombinant Plasmid Construction and Protein Expression-Purification Results

Schematic diagrams of the six protein sequences are shown in Figure 1A. The recombinant plasmids pET30a-CARDS, pET30a-P1, pET30a-P40-90, pET30a-P30, pET30a-P116, and pET30a-MPN133 were digested with NdeI and BamHI. Analysis of the digestion products by 1% agarose gel electrophoresis revealed the presence of expected fragments the vector fragment was approximately 5251 bp and the target gene fragments were 1848 bp, 4623 bp, 3336 bp, 774 bp, 2823 bp, and 870 bp, respectively, which were consistent with the expected sizes (Figure 1B). The correct recombinant plasmids were transformed into the expression strain BL21 (DE3) and single colonies were selected for small-scale induced expression. Results indicated that after induction, the bacterial culture expressed proteins with molecular weights of 70 kDa, 167 kDa, 118 kDa, 27 kDa, 105 kDa, and 32 kDa, which matched the expected molecular weights of the recombinant proteins CARDS, P1, P40-90, P30, P116 and MPN133. Electrophoretic analysis of the supernatant and pellet from the homogenized sample revealed that the antigens CARDS, P1, P40-90, P116 and MPN133 were expressed in a soluble form, while the antigen P30 was expressed as inclusion bodies. Protein purification was performed accordingly. P30 was purified using the inclusion body purification protocol; other antigens were purified by nickel ion affinity chromatography. Eluates with high purity were collected, subjected to buffer exchange and concentration, resulting in purified target proteins suitable for subsequent experiments (Figure 1C).

3.2. Specific Antibody Detection

After primary immunization, all recombinant proteins (CARDS, P1, P40-90, P30, P116, MPN133) induced the production of antigen specific antibodies in mice. Among them, sera from the CARDS-immunized group, diluted between 1000-fold and 32,000-fold, exhibited significantly higher OD_450_ values than sera from the other protein-immunized groups (Figure 2A). After boost immunization, the antigen specific antibody levels in the mouse serum were significantly increased compared with those after primary immunization. When diluted 1000-fold, the OD_450_ values of all protein-immunized groups were greater than 1.5, except for the P40-90 group (Figure 2B). The results of MP-specific antibody level detection showed that the antibodies induced by recombinant protein immunization could specifically bind to MP. The geometric mean titer (GMT) of serum antibodies in all groups ranged from 800 to 3200. Among them, the P30 group had the highest antibody titer, which was statistically different from the CARDS, P1 and MPN133 groups (Figure 2C).

3.3. Detection Results of Metabolic Inhibitory Antibodies

The serum sample of each group was obtained by mixing equal volumes of serum from six mice in the group, as the serum volume from a single mouse was less than 100 μL, which was insufficient to meet the requirements of repeated experiments. All experimental data were derived from three independent repeated experiments. Results of the MP metabolic inhibitory antibody assay showed that, compared with the MP growth control group, serum from the CARDS, P1, P40-90, P30, P116, MPN133 protein-immunized groups and the PBS control group all inhibited MP growth after 4-fold dilution. The mechanism by which MP inhibited by PBS group serum was that the addition of serum altered the pH value, disrupting the optimal growth environment for MP. And this effect was more pronounced at low serum dilutions. However, at a 2-fold dilution, although both immunized and PBS control sera exhibited growth inhibition, their degrees of inhibition differed: the OD_560_ values of the protein-immunized groups were all higher than those of the PBS control group, indicating that the serum from the protein-immunized groups indeed contained antibodies that inhibited MP growth. Among the protein-immunized groups, serum from the CARDS immunized group still inhibited MP growth after 16-fold dilution, serum from the P1 immunized group inhibited MP growth after 8-fold dilution, serum from the P116 immunized group inhibited MP growth after 256-fold dilution, and serum from the MPN133 immunized group inhibited MP growth after 32-fold dilution. These findings demonstrate that immunization with these antigens induced antibodies with potent MP growth-inhibitory activity (Figure 3A). Furthermore, the OD_560_ values were fitted with prepared MP content percentages (as a standard curve), yielding the logarithmic equation y = −0.556 ln(x) − 0.7149 (R^2^ = 0.8883). This indicates a negative correlation between MP content and OD_560_ values within a specific time period. An OD_560_ value corresponding to 50% MP content was calculated as 0.0964. Therefore, an OD_560_ value greater than 0.0964 in the experimental groups indicated an MP inhibition rate exceeding 50% (Figure 3B,C).

3.4. Pathological Changes of Lung Tissue

The lung tissues of mice were collected for histopathological analysis. The pathological score of the P1 group was higher than that of the PBS group, although this difference did not reach statistical significance (n = 6, p = 0.8133); the pathological scores of the P40-90 and P30 groups were lower than that of the PBS group, with no statistically significant differences (n = 6, p > 0.25); the pathological scores of the CARDS group, P116 group and MPN133 group were significantly lower than that of the PBS group (n = 6, p < 0.01) (Figure 4A,B). Notably, CARDS, P116 and MPN133 also showed good performance in the metabolic inhibition antibody assay.

4. Discussion

Since the 1960s, researchers have made preliminary explorations into MP vaccines, including the development of inactivated vaccines and attenuated live vaccines [17,26]. However, early clinical trials revealed that individuals vaccinated with inactivated vaccines exhibited more severe clinical symptoms upon MP infection than those in the placebo group. Attenuated live vaccines, due to the potential risk of virulence reversion, have not been able to enter clinical application [27,28,29]. In recent years, vaccine development strategies have shifted from traditional inactivated or attenuated vaccines to safer, more precise subunit and nucleic acid vaccines. Although these new vaccines show promising prospects in theory, their protective effects have proven suboptimal in practical research [30]. Currently, the challenges of MP vaccine research primarily include two aspects: first, inducing a durable and effective immune response while ensuring vaccine stability; and second, ensuring vaccine safety [31,32]. Consequently, selecting more suitable vaccine targets and adopting new immunization strategies will be the key focus of future research.

Therefore, in this study, recombinant proteins, CARDS, P1, P40-90, P30, P116 and MPN133, were produced and evaluated for their immunogenicity and protection against MP infection through animal experiments, aiming to facilitate the development of MP vaccine. The immunogenicity analysis results showed that CARDS, P1, P40-90, P30, P116 and MPN133 all induced high levels specific antibodies, and the antibody levels increased with the number of immunizations. Furthermore, after the primary immunization, protein CARDS generated a stronger antibody response compared to the other five proteins, indicating its different immune characteristics. The MP metabolic inhibition antibody assay revealed that after the boost immunization, the serum from CARDS, P1, P40-90, P30, P116 and MPN133 group effectively inhibited MP growth. Additionally, CARDS, P116 and MPN133 exhibited a stronger ability to induce inhibitory antibodies, especially P116 and MPN133. The lung pathological examination results showed that, except for the P1 group, the pathological scores of the protein-immunized groups were lower than those of the PBS group. Among them, the CARDS, P116 and MPN133 groups had significantly lower pathological scores compared to the PBS group. As the major adhesin protein of MP, P1 exhibited potential risk of vaccine-enhanced disease (VED) in this study. The underlying reason might be the functional bias in the antibody response induced by P1. Specifically, the antibodies elicited by P1 are only capable of binding to the surface epitopes of MP, while lacking neutralizing or bactericidal activity. On the contrary, these antibodies may exacerbate pulmonary inflammatory damage via the antibody-dependent enhancement (ADE) effect or by disrupting the local immune homeostasis in the host lung. This suggests that only when sufficient bacteriostatic antibodies targeting the functional epitopes of MP are induced can the infection process be effectively blocked and pathological damage be alleviated. Based on the comprehensive analysis of the above experimental data, we draw the following conclusion: among the six proteins, CARDS, P116 and MPN133 exhibit greater potential for MP vaccine development.

It is worth emphasizing that through this study, we have come to a profound realization that the core pathological feature of Mycoplasma pneumoniae (MP) infection is a pulmonary localized disease. This inherent characteristic dictates the research and evaluation logic for MP vaccines, which must move beyond the single framework centered on systemic immune indicators (e.g., serum antibody titers). While high serum antibody titers can reflect the activation level of the body’s immune response, they struggle to cross the pulmonary barrier and accumulate effectively at local infection sites such as the bronchi and pulmonary interstitium; their concentration is often far from the effective level required for local bactericidal activity. Therefore, future MP vaccine research should prioritize a focus on pulmonary-targeted immune responses and mucosal immunity.

While administration of the three candidate proteins (CARDS, P116, and MPN133) mitigated pathological lung damage in MP infected mice, they failed to confer complete protection against MP infection. This outcome underscores the significant challenges remaining in the development of efficacious MP vaccines. Substantial advancements beyond the current research foundation are imperative to overcome existing bottlenecks. The following strategies may provide effective directions for advancing the vaccine development process: Protein Optimization via Genetic Engineering: Conduct targeted modification of the original proteins through genetic engineering techniques (such as optimizing antigen epitope exposure and adding mucosal targeting sequences) to enhance the immunogenicity of the proteins and their retention capacity in local lung tissues. Synergistic Protein Combinations: Innovatively combine proteins with different immune characteristics (such as combining adhesion-related proteins with immune regulation-related proteins) to achieve a synergistic effect of “blocking pathogen adhesion + enhancing local immune response”. Multi-functional Fusion Protein Design: Construct fusion proteins that integrate multiple functional antigen fragments to simultaneously activate multiple defense mechanisms including humoral immunity, cellular immunity, and mucosal immunity. Implementing these strategies holds the potential to significantly augment the immunogenicity and overall protective efficacy of MP vaccines. The ultimate goal is to develop novel vaccines precisely tailored to the immunological profile of localized MP infection, thereby achieving potent and durable protection.

5. Conclusions

This study successfully expressed six key MP proteins (CARDS, P1, P40-90, P30, P116, MPN133) using an Escherichia coli expression system and systematically evaluated their immunogenicity and immune reactivity in a mouse model. The results showed that all six proteins exhibited good immunogenicity; among them, CARDS, P116 and MPN133 not only induced strong inhibitory antibodies but also significantly alleviated lung pathological damage in MP-infected mice, demonstrating high potential for vaccine development and laying a foundation for subsequent research of MP vaccine. However, these antigens have clear limitations: they failed to confer complete protection against MP infection, and their induced serum antibodies struggle to cross the pulmonary barrier for effective local enrichment, while lacking optimization for lung-targeted mucosal immunity. These findings lay a targeted foundation for subsequent MP vaccine research.