{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,4]],"date-time":"2026-11-04T23:44:26Z","timestamp":1793835866898,"version":"4.6.1"},"reference-count":25,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Mycoplasma pneumoniae (MP) is a major cause of respiratory tract infections in children and adolescents. Currently, there is no licensed vaccine, underscoring the urgent need for the development of safe and effective vaccines. Objective: The aim of this study is to develop a recombinant subunit vaccine candidate incorporating three antigens: the P1 protein, the P40\/90 complex, and a detoxified mutant of community-acquired respiratory distress syndrome toxin. The protective efficacy of this vaccine candidate was also evaluated. Methods: Target genes were codon-optimized for expression in E. coli, and the recombinant proteins were successfully expressed and purified. The low-toxicity CARDS toxin mutant was screened based on TNF-\u03b1 secretion levels in stimulated RAW264.7 cells. A three-component vaccine composed of P1, P40\/90, and the mutant CARDS toxin was formulated and adjuvanted with either Al(OH)3 alone or in combination with CpG. Mice were immunized, and immunogenicity was assessed by measuring antigen-specific IgG antibody titers. Protective efficacy was evaluated following challenge by analyzing lung histopathology, bacterial load, and inflammatory cytokine levels. Results: Seven high-purity recombinant proteins were successfully produced, including P1, the P40\/90 complex, wild-type CARDS toxin, and four CARDS toxin mutants (E132A, E132Q, H36A, R10A). The E132A mutant was selected due to its significantly reduced toxicity while retaining immunogenicity. The three-component vaccine effectively elicited antibody responses against each of the included antigens. After three immunizations, IgG antibody titers in all groups reached approximately 104. Immunized mice showed markedly reduced pulmonary pathology scores (control group: 2 or 2.67; immunized groups: 1.67, 1.33, and 0) and significantly decreased bacterial loads in lung tissue (control: 30.11 \u00b1 10.40 cp\/\u03bcL; immunized groups: 20.72 \u00b1 4.37 cp\/\u03bcL and 8.51 \u00b1 8.32 cp\/\u03bcL). Furthermore, the group receiving the alum + CpG adjuvant exhibited approximately a 10-fold higher antibody response compared with the alum-only group, indicating enhanced protective efficacy. Conclusions: The three-component candidate vaccine, MPtriV, adjuvanted with Al(OH)3 + CpG, demonstrates promising immunogenicity, safety, and protective efficacy against Mycoplasma pneumoniae infection, providing a viable strategy and experimental foundation for the development of MP subunit vaccines.<\/jats:p>","DOI":"10.3390\/vaccines14040330","type":"journal-article","created":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T07:25:12Z","timestamp":1775633112000},"page":"330","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Three-Component Subunit Vaccine Induces Protective Immunity Against Mycoplasma pneumoniae in Mice"],"prefix":"10.3390","volume":"14","author":[{"given":"Han","family":"Wang","sequence":"first","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiantian","family":"Wang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuoran","family":"Hou","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruixi","family":"Liu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianhui","family":"Liu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhu","family":"Zhou","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"School of Life Sciences and Medical Enginering, Anhui University, Hefei 230601, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuchen","family":"Hou","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Li","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Wu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6655-1866","authenticated-orcid":false,"given":"Bo","family":"Liu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"001910","DOI":"10.1099\/jmm.0.001910","article-title":"Mycoplasma pneumoniae: Not a Typical Respiratory Pathogen","volume":"73","author":"Rowlands","year":"2024","journal-title":"J. Med. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Bongiovanni, M. (2026). From Respiratory Pathogen to Systemic Threat: Rethinking Mycoplasma pneumoniae Infections. Microorganisms, 14.","DOI":"10.3390\/microorganisms14020419"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e454","DOI":"10.1097\/INF.0000000000004946","article-title":"Mycoplasma pneumoniae: Extrapulmonary Manifestations in Children with Focus on Mucocutaneous Disease","volume":"44","author":"Ramien","year":"2025","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e00255-25","DOI":"10.1128\/spectrum.00255-25","article-title":"Therapeutic Efficacy of Lascufloxacin in Patients with Mycoplasma pneumoniae Pneumonia","volume":"13","author":"Miyashita","year":"2025","journal-title":"Microbiol. Spectr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"271","DOI":"10.3892\/etm.2024.12559","article-title":"Exploring the Pathogenetic Mechanisms of Mycoplasma pneumoniae (Review)","volume":"28","author":"Georgakopoulou","year":"2024","journal-title":"Exp. Ther. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e02186-14","DOI":"10.1128\/mBio.02186-14","article-title":"ADP-Ribosylation of NLRP3 by Mycoplasma pneumoniae CARDS Toxin Regulates Inflammasome Activity","volume":"5","author":"Bose","year":"2014","journal-title":"mBio"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1007\/s00284-022-03103-0","article-title":"Insight into the Pathogenic Mechanism of Mycoplasma pneumoniae","volume":"80","author":"Hu","year":"2023","journal-title":"Curr. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5188","DOI":"10.1038\/s41467-020-18777-y","article-title":"Immunodominant Proteins P1 and P40\/P90 from Human Pathogen Mycoplasma pneumoniae","volume":"11","author":"Vizarraga","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Jiang, Z., Li, S., Zhu, C., Zhou, R., and Leung, P.H.M. (2021). Mycoplasma pneumoniae Infections: Pathogenesis and Vaccine Development. Pathogens, 10.","DOI":"10.3390\/pathogens10020119"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5165","DOI":"10.1073\/pnas.1420308112","article-title":"Structure of CARDS Toxin, a Unique ADP-Ribosylating and Vacuolating Cytotoxin from Mycoplasma pneumoniae","volume":"112","author":"Becker","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1186\/s10020-019-0105-2","article-title":"High Co-Expression of TNF-\u03b1 and CARDS Toxin Is a Good Predictor for Refractory Mycoplasma pneumoniae Pneumonia","volume":"25","author":"Li","year":"2019","journal-title":"Mol. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1111\/mmi.12680","article-title":"Functional Mapping of Community-acquired Respiratory Distress Syndrome (CARDS) Toxin of Mycoplasma pneumoniae Defines Regions with ADP-ribosyltransferase, Vacuolating and Receptor-binding Activities","volume":"93","author":"Kannan","year":"2014","journal-title":"Mol. Microbiol."},{"key":"ref_13","first-page":"597","article-title":"Efficacy of Inactivated Mycoplasma pneumoniae Vaccine in Man","volume":"28","author":"Mogabgab","year":"1975","journal-title":"Dev. Biol. Stand."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.etap.2017.01.001","article-title":"The Effect of Platycodin D on the Expression of Cytoadherence Proteins P1 and P30 in Mycoplasma pneumoniae Models","volume":"49","author":"Meng","year":"2017","journal-title":"Environ. Toxicol. Pharmacol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6724","DOI":"10.1073\/pnas.0510644103","article-title":"ADP-Ribosylating and Vacuolating Cytotoxin of Mycoplasma pneumoniae Represents Unique Virulence Determinant among Bacterial Pathogens","volume":"103","author":"Kannan","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"690","DOI":"10.2741\/2093","article-title":"Adhesion Proteins of Mycoplasma pneumoniae","volume":"12","author":"Chaudhry","year":"2007","journal-title":"Front. Biosci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"592","DOI":"10.2174\/0929866523666160502155414","article-title":"Designing, Expression and Immunological Characterization of a Chimeric Protein of Mycoplasma pneumoniae","volume":"23","author":"Chen","year":"2016","journal-title":"PPL"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1139\/w2012-041","article-title":"Protective Immune Responses in Mice Induced by Intramuscular and Intranasal Immunization with a Mycoplasma pneumoniae P1C DNA Vaccine","volume":"58","author":"Zhu","year":"2012","journal-title":"Can. J. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.eng.2021.06.012","article-title":"A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses","volume":"13","author":"Liu","year":"2022","journal-title":"Engineering"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1128\/CMR.00114-16","article-title":"Mycoplasma pneumoniae from the Respiratory Tract and Beyond","volume":"30","author":"Waites","year":"2017","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1016\/j.ijmm.2015.08.021","article-title":"New Insights in the Outbreak Pattern of Mycoplasma pneumoniae","volume":"305","author":"Jacobs","year":"2015","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2482702","DOI":"10.1080\/22221751.2025.2482702","article-title":"Rational Mpox Vaccine Design: Immunogenicity and Protective Effect of Individual and Multicomponent Proteins in Mice","volume":"14","author":"Cheng","year":"2025","journal-title":"Emerg. Microbes Infect."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"111817","DOI":"10.1016\/j.intimp.2024.111817","article-title":"Evaluation of Different Types of Adjuvants in a Malaria Transmission-Blocking Vaccine","volume":"131","author":"Yu","year":"2024","journal-title":"Int. Immunopharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"122569","DOI":"10.1016\/j.biomaterials.2024.122569","article-title":"The Orientation of CpG Conjugation on Aluminum Oxyhydroxide Nanoparticles Determines the Immunostimulatory Effects of Combination Adjuvants","volume":"308","author":"Liang","year":"2024","journal-title":"Biomaterials"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.micpath.2013.07.004","article-title":"Development of Protective Anti-Mycoplasma pneumoniae Antibodies after Immunization of Guinea Pigs with the Combination of a P1-P30 Chimeric Recombinant Protein and Chitosan","volume":"64","author":"Hausner","year":"2013","journal-title":"Microb. Pathog."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/4\/330\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T10:17:54Z","timestamp":1775643474000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/4\/330"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,7]]},"references-count":25,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["vaccines14040330"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14040330","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,7]]}}}