{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T15:35:04Z","timestamp":1784043304548,"version":"3.55.0"},"reference-count":27,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T00:00:00Z","timestamp":1749168000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Higher Education, Malaysia","award":["MO001-2021"],"award-info":[{"award-number":["MO001-2021"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: The emergence of multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) as a leading cause of fatal hospital-acquired infections underscores the urgent need for effective vaccines. While oral vaccines using live Bacillus subtilis spores expressing A. baumannii TonB-dependent receptor (TBDR) show promise, biosafety concerns regarding recombinant spore persistence necessitate alternative strategies. Here, we evaluated chemically inactivated B. subtilis spores displaying TBDR as a safer yet immunogenic vaccine candidate. Methods: Recombinant spores were inactivated using iron-ethanol sporicidal solution and administered to BALB\/c mice (8\u201312 weeks old) to assess safety and immunogenicity. Toxicity was evaluated through clinical monitoring, serum biochemistry, and histopathology. Immune responses were characterized by T\/B cell activation, IgG\/IgA titers, and mucosal sIgA levels. Protective efficacy was determined by challenging immunized mice with MDR A. baumannii Ab35 and quantifying bacterial loads and examining tissue pathology. Results: The inactivated spores exhibited an excellent safety profile, with no adverse effects on clinical parameters, organ function, or tissue integrity. Immunization induced robust systemic and mucosal immunity, evidenced by elevated CD4+\/CD8+ T cells, B cells, and antigen-specific IgG\/IgA in serum and mucosal secretions. Following the challenge, vaccinated mice showed significantly reduced pulmonary bacterial burdens (&gt;90% reduction), and preserved lung and spleen architecture compared to controls, which developed severe inflammation and tissue damage. Conclusions: These findings demonstrate that inactivated B. subtilis spores expressing TBDR are a safe, orally administrable vaccine platform that elicits protective immunity against MDR A. baumannii. By addressing biosafety concerns associated with live spores while maintaining efficacy, this approach represents a critical advance toward preventing high-risk nosocomial infections.<\/jats:p>","DOI":"10.3390\/vaccines13060616","type":"journal-article","created":{"date-parts":[[2025,6,6]],"date-time":"2025-06-06T09:43:14Z","timestamp":1749202994000},"page":"616","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Immunization with Inactivated Bacillus subtilis Spores Expressing TonB-Dependent Receptor (TBDR) Protects Against Multidrug-Resistant Acinetobacter baumannii Infection"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3869-2383","authenticated-orcid":false,"given":"Amalia A.","family":"Saperi","sequence":"first","affiliation":[{"name":"Tropical Infectious Diseases Research and Education Centre (TIDREC), University Malaya,  Kuala Lumpur 50603, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atiqah","family":"Hazan","sequence":"additional","affiliation":[{"name":"Tropical Infectious Diseases Research and Education Centre (TIDREC), University Malaya,  Kuala Lumpur 50603, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nurfatihah","family":"Zulkifli","sequence":"additional","affiliation":[{"name":"Tropical Infectious Diseases Research and Education Centre (TIDREC), University Malaya,  Kuala Lumpur 50603, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6322-6718","authenticated-orcid":false,"given":"Hai-Yen","family":"Lee","sequence":"additional","affiliation":[{"name":"Tropical Infectious Diseases Research and Education Centre (TIDREC), University Malaya,  Kuala Lumpur 50603, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5953-2886","authenticated-orcid":false,"given":"Nor-Aziyah","family":"MatRahim","sequence":"additional","affiliation":[{"name":"Institute for Medical Research (IMR), Setia Alam,  Shah Alam 40170, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9267-1420","authenticated-orcid":false,"given":"Sazaly","family":"AbuBakar","sequence":"additional","affiliation":[{"name":"Tropical Infectious Diseases Research and Education Centre (TIDREC), University Malaya,  Kuala Lumpur 50603, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Lee, C.R., Lee, J.H., Park, M., Park, K.S., Bae, I.K., Kim, Y.B., Cha, C.J., Jeong, B.C., and Lee, S.H. (2017). Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options. Front. Cell. Infect. Microbiol., 7.","DOI":"10.3389\/fcimb.2017.00055"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Moubareck, C.A., and Halat, D.H. (2020). Insights into Acinetobacter baumannii: A Review of Microbiological, Virulence, and Resistance Traits in a Threatening Nosocomial Pathogen. Antibiotics, 9.","DOI":"10.3390\/antibiotics9030119"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"243","DOI":"10.4161\/viru.19700","article-title":"Acinetobacter baumannii: An Emerging Opportunistic Pathogen","volume":"3","author":"Howard","year":"2012","journal-title":"Virulence"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Elbehiry, A., Marzouk, E., Moussa, I., Mushayt, Y., Algarni, A.A., Alrashed, O.A., Alghamdi, K.S., Almutairi, N.A., Anagreyyah, S.A., and Alzahrani, A. (2023). The Prevalence of Multidrug-Resistant Acinetobacter baumannii and Its Vaccination Status among Healthcare Providers. Vaccines, 11.","DOI":"10.3390\/vaccines11071171"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Ma, C., and McClean, S. (2021). Mapping Global Prevalence of Acinetobacter baumannii and Recent Vaccine Development to Tackle It. Vaccines, 9.","DOI":"10.3390\/vaccines9060570"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3784","DOI":"10.1080\/21645515.2021.1927412","article-title":"Facing the Challenges of Multidrug-Resistant Acinetobacter baumannii: Progress and Prospects in the Vaccine Development","volume":"17","author":"Lee","year":"2021","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Tan, Y.C., and Lahiri, C. (2022). Promising Acinetobacter baumannii Vaccine Candidates and Drug Targets in Recent Years. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.900509"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"179","DOI":"10.4014\/jmb.1807.06066","article-title":"Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications","volume":"29","author":"Zhang","year":"2019","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1186\/s12934-020-01436-8","article-title":"Bacillus subtilis: A Universal Cell Factory for Industry, Agriculture, Biomaterials and Medicine","volume":"19","author":"Su","year":"2020","journal-title":"Microb. Cell Fact."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12602-022-09909-5","article-title":"Bacillus Species as Direct-Fed Microbial Antibiotic Alternatives for Monogastric Production","volume":"15","author":"Bahaddad","year":"2023","journal-title":"Probiotics Antimicrob. Proteins"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/j.jsps.2022.03.010","article-title":"A Guide to Oral Vaccination: Highlighting Electrospraying as a Promising Manufacturing Technique toward a Successful Oral Vaccine Development","volume":"30","author":"Aldossary","year":"2022","journal-title":"Saudi Pharm. J."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"De Souza, R.D., Batista, M.T., Luiz, W.B., Cavalcante, R.C.M., Amorim, J.H., Bizerra, R.S.P., Martins, E.G., and De Souza Ferreira, L.C. (2014). Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0087454"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Sung, J.C.C., Lai, N.C.Y., Wu, K.C., Choi, M.C., Ma, C.H.Y., Lin, J., Kuok, C.N., Leong, W.L., Lam, W.K., and Hamied, Y.K. (2022). Safety and Immunogenicity of Inactivated Bacillus subtilis Spores as a Heterologous Antibody Booster for COVID-19 Vaccines. Vaccines, 10.","DOI":"10.3390\/vaccines10071014"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1146\/annurev.micro.112408.134247","article-title":"TonB-Dependent Transporters: Regulation, Structure, and Function","volume":"64","author":"Noinaj","year":"2010","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"MatRahim, N.A., Jones, K.M., Keegan, B.P., Strych, U., Zhan, B., Lee, H.Y., and AbuBakar, S. (2023). TonB-Dependent Receptor Protein Displayed on Spores of Bacillus subtilis Stimulates Protective Immune Responses against Acinetobacter baumannii. Vaccines, 11.","DOI":"10.3390\/vaccines11061106"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1038\/s41392-023-01471-y","article-title":"T Cells in Health and Disease","volume":"8","author":"Sun","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Schijns, V., Majhen, D., Van Der Ley, P., Thakur, A., Summerfield, A., Berisio, R., Nativi, C., Fern\u00e1ndez-Tejada, A., \u00c1lverez-Dominguez, C., and Gizurarson, S. (2021). Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13040501"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.coi.2014.02.003","article-title":"Novel Vaccine Approaches for Protection against Intracellular Pathogens","volume":"28","author":"Griffiths","year":"2014","journal-title":"Curr. Opin. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1538","DOI":"10.1002\/eji.200636875","article-title":"Bacillus subtilis Spores: A Novel Microparticle Adjuvant Which Can Instruct Balanced Th1 and Th2 Immune Response to Specific Antigen","volume":"37","author":"Barnes","year":"2007","journal-title":"Eur. J. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3266","DOI":"10.1016\/j.vaccine.2012.03.016","article-title":"Killed Bacillus subtilis Spores as a Mucosal Adjuvant for an H5N1 Vaccine","volume":"30","author":"Song","year":"2012","journal-title":"Vaccine"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"979","DOI":"10.4161\/hv.20694","article-title":"Bacillus subtilis: A Temperature Resistant and Needle Free Delivery System of Immunogens","volume":"8","author":"Amuguni","year":"2012","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Afinjuomo, F., Abdella, S., Youssef, S.H., Song, Y., and Garg, S. (2021). Inulin and Its Application in Drug Delivery. Pharmaceuticals, 14.","DOI":"10.3390\/ph14090855"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1016\/j.carbpol.2015.08.022","article-title":"Inulin, a Flexible Oligosaccharide. II: Review of Its Pharmaceutical Applications","volume":"134","author":"Mensink","year":"2015","journal-title":"Carbohydr. Polym."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1728","DOI":"10.1111\/jgh.16619","article-title":"Short-Chain Fatty Acids: Bridges between Diet, Gut Microbiota, and Health","volume":"39","author":"Wang","year":"2024","journal-title":"J. Gastroenterol. Hepatol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.ebiom.2016.11.015","article-title":"Advax, a Delta Inulin Microparticle, Potentiates In-Built Adjuvant Property of Co-Administered Vaccines","volume":"15","author":"Hayashi","year":"2017","journal-title":"EBioMedicine"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Counoupas, C., Pinto, R., Nagalingam, G., Britton, W.J., Petrovsky, N., and Triccas, J.A. (2017). Delta Inulin-Based Adjuvants Promote the Generation of Polyfunctional CD4+ T Cell Responses and Protection against Mycobacterium tuberculosis Infection. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-09119-y"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1038\/s41385-022-00578-9","article-title":"Mucosal Immunization with a Delta-Inulin Adjuvanted Recombinant Spike Vaccine Elicits Lung-Resident Immune Memory and Protects Mice against SARS-CoV-2","volume":"15","author":"Stewart","year":"2022","journal-title":"Mucosal Immunol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/6\/616\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:47:49Z","timestamp":1760032069000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/6\/616"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,6]]},"references-count":27,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["vaccines13060616"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13060616","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,6]]}}}