{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T13:39:52Z","timestamp":1774013992162,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T00:00:00Z","timestamp":1773964800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2023YFC2606001"],"award-info":[{"award-number":["2023YFC2606001"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: The global spread of monkeypox virus (MPXV) highlights an urgent need for thermostable and easily administrable vaccines. Current orthopoxvirus vaccines are limited by cold-chain dependence and inconvenient injection-based delivery. Objectives: This study aimed to develop a dissolvable microneedle (DMN) vaccine against monkeypox based on a replication-deficient orthopoxvirus platform, through systematic formulation screening, stabilization mechanism exploration, and rigorous in vivo immunogenicity evaluation. Methods: A film-based approach was adopted for efficient, high-throughput formulation screening and thermostability assessment. NTV was mixed with excipients and dried into solid films. Stability was monitored via RT-qPCR after storage at 4 \u00b0C to 40 \u00b0C. The lead formulation was physically characterized, then used to fabricate MVA-BN-loaded DMN patches, which were further evaluated for in vivo immunogenicity via immunization in BALB\/c mice. Results: The optimal formulation F2 (containing dextran, L-threonine, and BSA\/HSA) showed a potency loss of only ~1 log10 after 2 months at 25 \u00b0C, and &lt;1 log10 loss after 1 week at 37 \u00b0C. SEM revealed a porous virus-entrapment morphology, and FTIR indicated enhanced hydrogen bonding between the virus and the dextran matrix. The formulation was successfully manufactured into DMNs that dissolved within 5 min. In mice, these DMNs elicited robust MPXV-specific IgG and neutralizing antibody responses, with immunogenicity comparable to that induced by conventional intramuscular injection. Conclusions: This study successfully established a thermostable formulation and dissolvable microneedle delivery platform for replication-deficient orthopoxvirus vaccines against monkeypox. The optimized DMN vaccine induced robust MPXV-specific immune responses in mice with immunogenicity comparable to intramuscular injection, addressing the core limitations of current vaccines and providing a promising solution for monkeypox prevention.<\/jats:p>","DOI":"10.3390\/vaccines14030276","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T10:49:58Z","timestamp":1774003798000},"page":"276","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Dissolvable Microneedle Delivery of a Replication-Deficient Orthopoxvirus Vaccine: Formulation Screening and Immunogenicity Evaluation for Monkeypox Prevention"],"prefix":"10.3390","volume":"14","author":[{"given":"Bin","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei 230032, China"},{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kehui","family":"Wang","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhiyao","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei 230032, China"},{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weihua","family":"Liu","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"},{"name":"Department of Health Statistics, School of Public Health, China Medical University, Shenyang 110122, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xianhuang","family":"Li","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linhao","family":"Li","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Renhui","family":"Zhou","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xingyue","family":"Du","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin","family":"Jin","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaqing","family":"Xu","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rihui","family":"Qin","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8152-0734","authenticated-orcid":false,"given":"Xiong","family":"Liu","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dayang","family":"Zou","sequence":"additional","affiliation":[{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei 230032, China"},{"name":"Chinese PLA Center for Disease Control and Prevention, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1126\/science.ade6360","article-title":"Structure of monkeypox virus DNA polymerase holoenzyme","volume":"379","author":"Peng","year":"2023","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1038\/s41594-023-01201-6","article-title":"Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores","volume":"31","author":"Datler","year":"2024","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"509","DOI":"10.15585\/mmwr.mm7114a1","article-title":"Monkeypox in a Traveler Returning from Nigeria\u2014Dallas, Texas, July 2021","volume":"71","author":"Rao","year":"2022","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1007\/s00705-023-05904-5","article-title":"Genomic characterization and molecular evolution of human monkeypox viruses","volume":"168","author":"Ferrareze","year":"2023","journal-title":"Arch. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1016\/j.jaad.2022.12.035","article-title":"A dermatologic assessment of 101 mpox (monkeypox) cases from 13 countries during the 2022 outbreak: Skin lesion morphology, clinical course, and scarring","volume":"88","author":"Prasad","year":"2023","journal-title":"J. Am. Acad. Dermatol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1126\/science.adg8116","article-title":"APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016","volume":"382","author":"Neher","year":"2023","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5376","DOI":"10.1038\/s41467-025-61134-0","article-title":"Monkeypox virus spreads from cell-to-cell and leads to neuronal death in human neural organoids","volume":"16","author":"Fahmi","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3265","DOI":"10.1038\/s41467-024-47328-y","article-title":"Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection","volume":"15","author":"Tamir","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1016\/S1473-3099(23)00057-9","article-title":"Effectiveness of one dose of MVA-BN smallpox vaccine against mpox in England using the case-coverage method: An observational study","volume":"23","author":"Bertran","year":"2023","journal-title":"Lancet Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"125515","DOI":"10.1016\/j.ijbiomac.2023.125515","article-title":"Immunogenic proteins and potential delivery platforms for mpox virus vaccine development: A rapid review","volume":"245","author":"Wang","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Garcia-Atutxa, I., Mondragon-Teran, P., Huerta-Saquero, A., and Villanueva-Flores, F. (2024). Advancements in monkeypox vaccines development: A critical review of emerging technologies. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1456060"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"101045","DOI":"10.1016\/j.lanmic.2024.101045","article-title":"Immunogenicity of MVA-BN vaccine deployed as mpox prophylaxis: A prospective, single-centre, cohort study and analysis of transcriptomic predictors of response","volume":"6","author":"Drennan","year":"2025","journal-title":"Lancet Microbe"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Srivastava, S., Kumar, S., Jain, S., Mohanty, A., Thapa, N., Poudel, P., Bhusal, K., Al-Qaim, Z.H., Barboza, J.J., and Padhi, B.K. (2023). The Global Monkeypox (Mpox) Outbreak: A Comprehensive Review. Vaccines, 11.","DOI":"10.3390\/vaccines11061093"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"105703","DOI":"10.1016\/j.ebiom.2025.105703","article-title":"Immunological analysis of LC16m8 vaccine: Preclinical and early clinical insights into mpox","volume":"115","author":"Kobiyama","year":"2025","journal-title":"eBioMedicine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2278900","DOI":"10.1080\/22221751.2023.2278900","article-title":"Non-replicating vaccinia virus NTV as an effective next-generation smallpox and monkeypox vaccine: Evidence from mouse and rhesus monkey models","volume":"12","author":"Chu","year":"2023","journal-title":"Emerg. Microbes Infect."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e96","DOI":"10.1016\/S2666-5247(21)00269-X","article-title":"Safety and dose-sparing effect of Japanese encephalitis vaccine administered by microneedle patch in uninfected, healthy adults (MNA-J): A randomised, partly blinded, active-controlled, phase 1 trial","volume":"3","author":"Iwata","year":"2022","journal-title":"Lancet Microbe"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e012247","DOI":"10.1136\/bmjgh-2023-012247","article-title":"Immunogenicity, safety, usability and acceptability of microarray patches for vaccination: A systematic review and meta-analysis","volume":"8","author":"Berger","year":"2023","journal-title":"BMJ Glob. Health"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1016\/j.jconrel.2022.05.045","article-title":"Dissolving microneedles: Applications and growing therapeutic potential","volume":"348","author":"Sartawi","year":"2022","journal-title":"J. Control. Release"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e2102595118","DOI":"10.1073\/pnas.2102595118","article-title":"Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity","volume":"118","author":"Caudill","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"114263","DOI":"10.1016\/j.jconrel.2025.114263","article-title":"Development of a dissolving microneedle patch for transdermal delivery of SARS-CoV-2 mRNA vaccine with enhanced stability and immunogenicity","volume":"388","author":"Tang","year":"2025","journal-title":"J. Control. Release"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e2302410","DOI":"10.1002\/adma.202302410","article-title":"Exploiting Unique Features of Microneedles to Modulate Immunity","volume":"35","author":"Edwards","year":"2023","journal-title":"Adv. Mater."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1879","DOI":"10.1016\/S0140-6736(24)00532-4","article-title":"A measles and rubella vaccine microneedle patch in The Gambia: A phase 1\/2, double-blind, double-dummy, randomised, active-controlled, age de-escalation trial","volume":"403","author":"Adigweme","year":"2024","journal-title":"Lancet"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Adediran, E., Arte, T., Pasupuleti, D., Vijayanand, S., Singh, R., Patel, P., Gulani, M., Ferguson, A., Uddin, M., and Zughaier, S.M. (2025). Delivery of PLGA-Loaded Influenza Vaccine Microparticles Using Dissolving Microneedles Induces a Robust Immune Response. Pharmaceutics, 17.","DOI":"10.3390\/pharmaceutics17040510"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1007\/s13346-023-01430-8","article-title":"The promise of microneedle technologies for drug delivery","volume":"14","author":"Donnelly","year":"2024","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.jconrel.2019.08.039","article-title":"Skin delivery of trivalent Sabin inactivated poliovirus vaccine using dissolvable microneedle patches induces neutralizing antibodies","volume":"311\u2013312","author":"Donadei","year":"2019","journal-title":"J. Control. Release"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1080\/21645515.2021.1887692","article-title":"Formulation Development and Improved Stability of a Combination Measles and Rubella Live-Viral Vaccine Dried for Use in the Nanopatch(TM) Microneedle Delivery System","volume":"17","author":"Wan","year":"2021","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2100095","DOI":"10.1002\/adtp.202100095","article-title":"Thermostability of Measles and Rubella Vaccines in a Microneedle Patch","volume":"4","author":"Joyce","year":"2021","journal-title":"Adv. Ther."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Choo, J.J.Y., McMillan, C.L.D., Fernando, G.J.P., Hall, R.A., Young, P.R., Hobson-Peters, J., and Muller, D.A. (2021). Developing a Stabilizing Formulation of a Live Chimeric Dengue Virus Vaccine Dry Coated on a High-Density Microarray Patch. Vaccines, 9.","DOI":"10.3390\/vaccines9111301"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1038\/s41541-022-00443-7","article-title":"Microneedle patch as a new platform to effectively deliver inactivated polio vaccine and inactivated rotavirus vaccine","volume":"7","author":"Moon","year":"2022","journal-title":"npj Vaccines"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"903","DOI":"10.1021\/acs.biomac.1c01345","article-title":"Dissolving Microneedle Delivery of a Prophylactic HPV Vaccine","volume":"23","author":"Ray","year":"2022","journal-title":"Biomacromolecules"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4953","DOI":"10.1021\/acsabm.1c00240","article-title":"Skin vaccination with dissolvable microneedle patches incorporating influenza neuraminidase and flagellin protein nanoparticles induces broad immune protection against multiple influenza viruses","volume":"4","author":"Wang","year":"2021","journal-title":"ACS Appl. Bio Mater."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"123182","DOI":"10.1016\/j.ijpharm.2023.123182","article-title":"Gonococcal microparticle vaccine in dissolving microneedles induced immunity and enhanced bacterial clearance in infected mice","volume":"642","author":"Bagwe","year":"2023","journal-title":"Int. J. Pharm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"121393","DOI":"10.1016\/j.ijpharm.2021.121393","article-title":"A dual-delivery platform for vaccination using antigen-loaded nanoparticles in dissolving microneedles","volume":"613","author":"Vijayanand","year":"2022","journal-title":"Int. J. Pharm."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1002\/btm2.10098","article-title":"Hepatitis B vaccination using a dissolvable microneedle patch is immunogenic in mice and rhesus macaques","volume":"3","author":"Kodani","year":"2018","journal-title":"Bioeng. Transl. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"694","DOI":"10.1016\/j.ijbiomac.2020.10.227","article-title":"Pullulan in biomedical research and development\u2014A review","volume":"166","author":"Singh","year":"2021","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"147829","DOI":"10.1016\/j.ijbiomac.2025.147829","article-title":"Preparation of iron dextran complex and its effect on iron deficiency anemia in rats","volume":"330","author":"He","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"eaau4819","DOI":"10.1126\/sciadv.aau4819","article-title":"Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature","volume":"6","author":"Bajrovic","year":"2020","journal-title":"Sci. Adv."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1370","DOI":"10.1038\/s41587-022-01381-4","article-title":"Generation of a live attenuated influenza A vaccine by proteolysis targeting","volume":"40","author":"Si","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2782","DOI":"10.1016\/j.ymthe.2021.05.018","article-title":"Live-attenuated RNA hybrid vaccine technology provides single-dose protection against Chikungunya virus","volume":"29","author":"Voigt","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Shang, Y., Li, L., Zhang, T., Luo, Q., Yu, Q., Zeng, Z., Li, L., Jia, M., Tang, G., and Fan, S. (2022). Quantitative regulation of the thermal stability of enveloped virus vaccines by surface charge engineering to prevent the self-aggregation of attachment glycoproteins. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1010564"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1016\/j.ijbiomac.2022.02.038","article-title":"Analysis of the structure and properties of dextran produced by Weissella confusa","volume":"204","author":"Du","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"7082","DOI":"10.1039\/D0BM01433H","article-title":"Thermostabilization of viruses via complex coacervation","volume":"8","author":"Mi","year":"2020","journal-title":"Biomater. Sci."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Kim, A.Y., Kim, H., Park, S.Y., Park, S.H., Kim, J.S., Park, J.W., Park, J.H., and Ko, Y.J. (2021). Development of a Potent Stabilizer for Long-Term Storage of Foot-and-Mouth Disease Vaccine Antigens. Vaccines, 9.","DOI":"10.3390\/vaccines9030252"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"102365","DOI":"10.1016\/j.cis.2021.102365","article-title":"Applications of serum albumins in delivery systems: Differences in interfacial behaviour and interacting abilities with polysaccharides","volume":"290","author":"Yang","year":"2021","journal-title":"Adv. Colloid Interface Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1038\/s41392-025-02259-y","article-title":"Safety and immunogenicity of an HIV vaccine trial with DNA prime and replicating vaccinia boost","volume":"10","author":"Liu","year":"2025","journal-title":"Signal Transduct. Target. Ther."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/3\/276\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T11:03:31Z","timestamp":1774004611000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/3\/276"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,20]]},"references-count":45,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["vaccines14030276"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14030276","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,20]]}}}