An mRNA Vaccine Targeting the C-Terminal Region of P1 Protein Induces an Immune Response and Protects Against Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae, a cell wall-deficient pathogen, primarily affects children and adolescents, causing Mycoplasma pneumoniae pneumonia (MPP). Following the relaxation of non-pharmaceutical interventions (NPIs) post COVID-19, there has been a global increase in MPP cases and macrolide-resistant strains. Vaccination against M. pneumoniae is being explored as a promising approach to reduce infections, limit antibiotic misuse, and prevent the emergence of drug-resistant variants. We developed an mRNA vaccine, mRNA-SP+P1, incorporating a eukaryotic signal peptide (tissue-type plasminogen activator signal peptide) fused to the C-terminal region of the P1 protein. Targeting amino acids 1288 to 1518 of the P1 protein, the vaccine was administered intramuscularly to BALB/c mice in a three-dose regimen. To evaluate immunogenicity, we quantified anti-P1 IgG antibody titers using enzyme-linked immunosorbent assays (ELISAs) and assessed cellular immune responses by analyzing effector memory T cell populations using flow cytometry. We also tested the functional activity of vaccine-induced sera for their ability to inhibit adhesion of the ATCC M129 strain to KMB17 cells. The vaccine's protective efficacy was assessed against the ATCC M129 strain and its cross-protection against the ST3-resistant strain. Transcriptomic analysis was conducted to investigate gene expression changes in peripheral blood, aiming to uncover mechanisms of immune modulation. The mRNA-SP+P1 vaccine induces P1 protein-specific IgG antibodies and an effector memory T-cell response in BALB/c mice. Adhesion inhibition assays demonstrated that serum from vaccinated mice attenuatesthe adhesion ability of ATCC M129 to KMB17 cells. Furthermore, three doses of the vaccine confer significant and long-lasting, though partial, protection against the ATCC M129 strain and partial cross-protection against the ST3 drug-resistant strain. Transcriptome analysis revealed significant gene expression changes in peripheral blood, confirming the vaccine's capacity to elicit an immune response from the molecular level. Our results indicate that the mRNA-SP+P1 vaccine appears to be an effective vaccine candidate against the prevalence of Mycoplasma pneumoniae.

Keywords: ATCC M129 strain; Mycoplasma pneumoniae; P1 protein; ST3 drug-resistant strain; mRNA vaccine.

Figure 1

Design and encapsulation of mRNA-SP+P1. (A) Diagram of the SP+P1 mRNA construct, illustrating a fusion protein composed of a tissue-type plasminogen activator signal peptide (SP) linked to the C-terminal region (amino acids 1288–1518) of the P1 protein. (B) Formaldehyde-denatured agarose gel electrophoresis assessed the integrity of SP+P1. (C) High-performance liquid chromatography evaluated the purity of SP+P1. (D) Preparation of mRNA-SP+P1 involved mixing mRNA in an acidic aqueous solution, injecting organic phase lipids, and extruding the mixture through a microfluidic chip. (E) Expression of the C-terminal P1 protein region (1288aa–1518aa) was achieved in HEK293T cells transfected with SP+P1 using Lipofectamine 3000 for 48 h. (F) Physicochemical properties of mRNA-SP+P1 are presented as mean ± SD. (G) A representative TEM image illustrates the morphology of mRNA-SP+P1, with a scale bar of 100 nm.

Figure 2

Humoral and effector memory T cell responses in mRNA-SP+P1-accinated mice. Female BALB/c mice received 20 μg of the mRNA-SP+P1 vaccine or served as controls (n = 5). Immunizations were administered intramuscularly on days 0, 14, and 28. Serum samples were collected on days 14, 28, 42, 56, and 70 post-initial immunization. (A) Immunization and sample collection timeline. (B,C) Body weight and temperature were monitored for seven days post-first immunization (n = 5). (D) The IgG antibody titers of P1 protein in the immunized group were evaluated by enzyme-linked immunosorbent assay (ELISA) (n = 5). Data are presented as mean ± SD. Statistical significance was determined using repeated measures ANOVA (n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E,F) P1 protein-specific CD4⁺ and CD8⁺ effector memory T cells in the spleen were analyzed using flow cytometry (n = 3). (G,H) The inhibitory effect of serum from vaccinated mice on ACTT M129 adhesion to KMB17 cells was evaluated (n = 3). Positive cells were identified by specific fluorescent markers for M. pneumoniae on KMB17 cell surfaces, while negative cells lacked these markers. The data in (E,F,H) are presented as mean ± SD. Statistical significance was determined using an unpaired t-test (n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Efficacy of mRNA-SP+P1 in protecting mice against ATCC M129 challenge. (A) Immunization, challenge, and sample collection timeline. (B,C) Percentage changes in body weight and temperature post-infection (n = 5). (D) IgG antibody titers specific to M. pneumoniae antigen P1 post-infection (n = 5). (E) Lung M. pneumoniae load post-infection (n = 5). (F) Lung pathology scores post-infection (n = 5). (G) Lung histology with H&E staining post-infection; scale bar = 50 µm. Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (B,C), Mann-Whitney test (D,F), or unpaired t-test (E) (n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

The protective effect of mRNA-SP+P1 against ATCC M129 in mice, as assessed 62 days post-booster immunization. (A) Immunization, challenge, and sample collection timeline. (B,C) Percentage changes in body weight and temperature post-infection (n = 5). (D) Titers of M. pneumoniae-specific P1 IgG antibodies post-infection (n = 5). (E) Lung M. pneumoniae load post-infection (n = 3). (F) Lung pathology scores post-infection (n = 3). (G) H&E-stained lung sections post-infection (n = 3), scale bar = 50 µm. Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA (B,C), Mann-Whitney test (D), or unpaired t-test (E,F) (n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Efficacy of mRNA-SP+P1 in protecting mice against a challenge with an ST3-resistant strain on day 42 post-primary immunization. (A) Immunization, challenge, and sample collection timeline. (B,C) Percentage changes in body weight and temperature post-infection (n = 5). (D) IgG antibody titers specific to M. pneumoniae antigen P1 post-infection (n = 5). (E) M. pneumoniae load in lung tissue post-infection (n = 5). (F) Lung pathological scores post-infection (n = 5). (G) Lung histopathology with H&E staining post-infection (n = 5). Scale bar = 50 µm. Data are presented as mean ± SD. Statistical significance was assessed using two-way ANOVA (B,C) or Mann-Whitney test for non-parametric data (D,F) or unpaired t-test (E) (n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Transcriptome analysis of whole blood stimulated by mRNA-SP+P1. (A) Volcano plot illustrating differentially expressed genes, with upregulated genes in red and downregulated genes in green. (B) Gene Ontology (GO) enrichment analysis of upregulated genes, where the vertical axis denotes the GO term and the horizontal axis indicates the Rich factor. Dot size represents the number of genes per GO term, and dot color signifies Q value ranges. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of upregulated genes, with the vertical axis displaying significantly enriched pathways and the horizontal axis showing the Rich factor. Dot color reflects q value ranges.