肺炎支原体（Mycoplasma pneumoniae, MP）是一种常见的呼吸道病原体，主要感染儿童及青少年，引起肺炎支原体肺炎（Mycoplasma pneumoniae pneumonia, MPP），对儿童及青少年的健康造成严重威胁。在SARS - CoV - 2疫情防控期间实施的非药物性干预措施（non - pharmaceutical interventions, NPIs）解除后，MP感染率显著回升。值得注意的是，该病原体对大环内酯类抗生素的耐药率持续攀升，这一现象对传统抗生素治疗方案构成严峻挑战。在此背景下，通过疫苗接种预防感染、降低抗生素使用需求，或可作为应对MP高感染率及高耐药率问题的有效干预措施。

基于系统文献分析，本研究选择MP P1蛋白C端序列（1288aa - 1518aa）作为疫苗靶点。为提高蛋白表达效率，本研究对编码序列进行了密码子优化，并引入真核信号肽序列以确保蛋白的有效分泌。随后通过体外转录（In vitro transcription，IVT）技术合成mRNA分子，并利用脂质纳米颗粒（Lipid nanoparticles，LNP）递送系统进行包封，最终成功制备mRNA - SP + P1疫苗。该疫苗制剂具有良好的物理特性：包封效率达到（92.25 ± 2.89）%，粒径分布均一（88.18 ± 3.13 nm），Zeta电位为（-3.72 ± 0.21）mV，透射电子显微镜显示其呈规则的球形或近球形形态。

本研究通过BALB/c小鼠模型评估mRNA - SP ＋ P1疫苗的免疫原性。通过间隔14天的三次肌肉注射免疫方案，该疫苗成功诱导了显著的体液免疫反应和效应记忆T细胞（Effector memory T cell，Tem）应答。体液免疫分析显示，初次免疫后第14天即可检测到抗P1蛋白特异性IgG抗体，滴度为1×10⁴ - 1×10⁵（GMT：55715.24），二次免疫后抗体滴度显著提升至1×10⁶左右（GMT：891443.78），第三次免疫后稳定在1×10⁶ - 1×10⁷水平（GMT：3104187.53）。长期监测结果显示，初次免疫后第42天至第70天期间，抗体滴度呈缓慢下降趋势，最终停留在1×10⁵ - 1×10⁶水平（GMT：776046.88）。黏附抑制实验证实，免疫血清能够有效阻断MP与KMB17细胞的黏附，提示所诱导抗体具有生物学功能。在细胞免疫应答方面，流式细胞术检测结果显示，免疫组小鼠脾脏中P1蛋白特异性的CD4⁺ Tem和CD8⁺ Tem细胞比例显著高于对照组，表明该疫苗可诱导持久的免疫记忆，为长期免疫保护奠定了基础。

为评估疫苗的保护效果，本研究采用了ATCC M129标准株和ST3型耐药株感染免疫组与对照组小鼠。结果显示，针对ATCC M129标准株的感染，该疫苗可显著缓解免疫组小鼠的体重下降程度及发热反应，同时降低其肺部MP载量，并显著改善其肺组织病理学损伤。值得注意的是，疫苗的保护效果在初次免疫后第90天仍能维持，证实其具有持久的免疫保护作用。在ST3型耐药株小鼠感染模型中，虽然疫苗对免疫组小鼠肺组织病理损伤的改善作用有限，但仍能显著降低其肺部MP载量，这一结果提示该疫苗可通过有效减少耐药株在宿主体内的定植，从而降低其传播风险。

为进一步探索疫苗的免疫机制，本研究通过转录组测序对免疫组与对照组小鼠的全血基因表达谱进行了系统性分析。结果显示，免疫组中有503个基因上调，190个基因下调，这些差异基因显著富集于抗原加工与呈递、B细胞受体信号以及Th1/Th2细胞分化等关键免疫调控通路。值得注意的是，TNFRSF13c及H2基因家族成员的显著上调表达，提示该疫苗可能通过协同调控黏膜免疫、体液免疫和细胞免疫等多重免疫途径，共同发挥免疫保护效应。这一发现为深入理解mRNA - SP ＋ P1疫苗的作用机制提供了重要的分子生物学依据。

综上所述，本研究成功构建了靶向MP P1蛋白C端的mRNA疫苗，证实其能够诱导高效的体液免疫应答和效应记忆T细胞增殖。在BALB/c小鼠模型中，该疫苗对ATCC M129及ST3型耐药株感染均展现出良好的保护效力。本研究不仅为MP感染的防控提供了新型候选疫苗策略，同时也为其他耐药性病原体疫苗的研发提供了重要技术参考。未来研究可进一步优化抗原设计、扩展动物模型，并探索多价疫苗的开发，以提升疫苗的广谱性和临床应用潜力。

Mycoplasma pneumoniae (MP) is a prevalent respiratory pathogen primarily affecting children and adolescents, causing Mycoplasma pneumoniae pneumonia (MPP). Following the relaxation of non-pharmaceutical interventions (NPIs) used to control the SARS - CoV - 2 outbreak, MP infection rates have surged. Concurrently, macrolide antibiotic resistance is escalating, complicating traditional treatment approaches. Preventing infections and reducing antibiotic demand through vaccination may effectively address the high infection and drug resistance rates of MP.

Through a systematic literature review, our research identified the C - terminal sequence (1288aa - 1518aa) of the MP P1 protein as a promising candidate for vaccine development due to its pivotal role in mediating MP attachment to host cells and its relatively high conservation. To enhance protein expression efficiency, codon optimization was performed on the coding sequence, and a eukaryotic signal peptide sequence was incorporated to facilitate proper protein secretion. Subsequently, mRNA molecules were synthesized using in vitro transcription (IVT) technology and enveloped within a lipid nanoparticle (LNP) delivery system. The resultant mRNA - SP + P1 vaccine formulation demonstrated favorable physical characteristics: high encapsulation efficiency (92.25 ± 2.89)%, uniform particle size distribution (88.18 ± 3.13 nm), slightly negative Zeta potential (-3.72 ± 0.21) mV, and electron microscopy revealed a regular spherical or near-spherical morphology.

Our research assessed the immunogenicity of the mRNA - SP + P1 vaccine in a BALB/c mouse model. Following a three-dose intramuscular immunization regimen with a 14 - day interval, the vaccine elicited robust humoral immunity and effector memory T cell (Tem) responses. Analysis of humoral immunity revealed the presence of specific IgG antibodies against the P1 protein (titer:1×10⁴ - 1×10⁵，GMT：55715.24) 14 days post primary immunization. Subsequent immunizations led to a significant increase in antibody titer to 1×10⁶（GMT：891443.78）after the second dose, stabilizing at 1×10⁶ - 1×10⁷（GMT：3104187.53）after the third dose. Longitudinal monitoring demonstrated a gradual decline in antibody titer from day 42 to day 70 post primary immunization, plateauing at 1×10⁵ - 1×10⁶（GMT：776046.88）.

Adhesion inhibition assays confirmed the ability of the immune serum to block MP adhesion to KMB17 cells, indicating functional antibodies were induced. Evaluation of cellular immune responses via flow cytometry revealed a notable increase in P1 protein-specific CD4⁺ Tem and CD8⁺ Tem cell proportions in the spleens of vaccinated mice compared to controls, suggesting the vaccine's capacity to establish enduring immune memory responses, thereby establishing a basis for sustained immune protection.

To assess the vaccine's protective efficacy, this study exposed both immunized and control groups of mice to the ATCC M129 standard strain and the drug-resistant ST3 - type strain. Results demonstrated that the vaccine effectively mitigated weight loss and fever in mice infected with the ATCC M129 strain, along with a significant reduction in MP load in the lungs and notable improvement in lung tissue pathology. Importantly, the vaccine's protective effects persisted on the 90th day after the first immunization, indicating durable immune memory. In the case of the drug-resistant ST3 - type strain, while the vaccine had limited impact on lung tissue pathology, it still significantly decreased MP load in the lungs. This outcome suggests that the vaccine can lower the transmission risk of the drug-resistant ST3 - type strain by reducing its colonization in the host.

To elucidate the immune mechanisms underlying the vaccine's effects, this study conducted a systematic analysis of whole - blood gene expression profiles in immunized and control mice using transcriptome sequencing. In the immunized group, 503 genes were upregulated and 190 were downregulated. These differentially expressed genes were significantly enriched in key immune regulatory pathways, including antigen processing and presentation, B cell receptor signaling, and Th1/Th2 cell differentiation. Notably, the upregulation of TNFRSF13c and H2 gene family members suggests that the vaccine may confer immune protection by regulating multiple pathways, encompassing mucosal, humoral, and cellular immunity. This finding provides a crucial molecular basis for understanding the mechanism of the mRNA - SP + P1 vaccine.

Our research successfully developed an mRNA vaccine targeting the C - terminus of the MP P1 protein, demonstrating its ability to elicit a robust humoral and effector - memory T - cell response. In the BALB/c mouse model, the vaccine showed effective protection against infections from both the ATCC M129 standard strain and the ST3 - type multidrug-resistant strain. These findings present a promising vaccine strategy for preventing and controlling MP infections and provide valuable technical references for developing vaccines against other drug - resistant pathogens. Future research should focus on optimizing antigen design, expanding animal models, and exploring multivalent vaccine development to enhance broad - spectrum efficacy and clinical applicability.