{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T19:33:20Z","timestamp":1788982400467,"version":"build-2803163510"},"reference-count":94,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,4,13]],"date-time":"2023-04-13T00:00:00Z","timestamp":1681344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002347","name":"BMBF","doi-asserted-by":"publisher","award":["13N11455"],"award-info":[{"award-number":["13N11455"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3\u2032,5\u2032)-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB\/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1\/Th17 response characterized by high secretion of IFN-\u03b3, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.<\/jats:p>","DOI":"10.3390\/pharmaceutics15041238","type":"journal-article","created":{"date-parts":[[2023,4,14]],"date-time":"2023-04-14T01:32:03Z","timestamp":1681435923000},"page":"1238","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity"],"prefix":"10.3390","volume":"15","author":[{"given":"Thomas","family":"Ebensen","sequence":"first","affiliation":[{"name":"Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Arntz","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2286-3416","authenticated-orcid":false,"given":"Kai","family":"Schulze","sequence":"additional","affiliation":[{"name":"Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Hanefeld","sequence":"additional","affiliation":[{"name":"Merck KGaA, Frankfurter Stra\u00dfe 250, 64293 Darmstadt, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos A.","family":"Guzm\u00e1n","sequence":"additional","affiliation":[{"name":"Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8685-5672","authenticated-orcid":false,"given":"Regina","family":"Scherlie\u00df","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1126\/science.1075173","article-title":"The intangible value of vaccination","volume":"297","author":"Rappuoli","year":"2002","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.it.2008.09.006","article-title":"New horizons in adjuvants for vaccine development","volume":"30","author":"Reed","year":"2009","journal-title":"Trends Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2155","DOI":"10.1001\/jama.298.18.2155","article-title":"Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States","volume":"298","author":"Roush","year":"2007","journal-title":"JAMA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.ijpharm.2008.04.036","article-title":"Vaccine adjuvant systems: Enhancing the efficacy of sub-unit protein antigens","volume":"364","author":"Perrie","year":"2008","journal-title":"Int. J. Pharm."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6711","DOI":"10.1016\/j.vaccine.2014.08.089","article-title":"The mucosal immune system for vaccine development","volume":"32","author":"Lamichhane","year":"2014","journal-title":"Vaccine"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.ijpharm.2013.02.031","article-title":"Inhaled proteins: Challenges and perspectives","volume":"447","author":"Depreter","year":"2013","journal-title":"Int. J. Pharm."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1016\/j.coi.2011.03.009","article-title":"Defending the mucosa: Adjuvant and carrier formulations for mucosal immunity","volume":"23","author":"Lawson","year":"2011","journal-title":"Curr. Opin. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1111\/apm.12351","article-title":"Mucosal vaccines: A paradigm shift in the development of mucosal adjuvants and delivery vehicles","volume":"123","author":"Srivastava","year":"2015","journal-title":"APMIS"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1146\/annurev-bioeng-071811-150054","article-title":"Mucosal vaccine design and delivery","volume":"14","author":"Woodrow","year":"2012","journal-title":"Annu. Rev. Biomed. Eng."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1016\/j.jconrel.2011.03.009","article-title":"Intranasal immunization with poly(gamma-glutamic acid) nanoparticles entrapping antigenic proteins can induce potent tumor immunity","volume":"152","author":"Matsuo","year":"2011","journal-title":"J. Control. Release"},{"key":"ref_11","first-page":"243","article-title":"A New Wave of Vaccines for Non-Communicable Diseases: What Are the Regulatory Challenges?","volume":"70","author":"Darrow","year":"2015","journal-title":"Food Drug Law. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"49","DOI":"10.4155\/ppa.15.38","article-title":"Recent advances in vaccine delivery","volume":"5","author":"Cordeiro","year":"2016","journal-title":"Pharm. Pat. Anal."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1038\/nrd1176","article-title":"Recent advances in the discovery and delivery of vaccine adjuvants","volume":"2","author":"Valiante","year":"2003","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1016\/j.drudis.2011.04.006","article-title":"Particulate vaccines: On the quest for optimal delivery and immune response","volume":"16","author":"Rejman","year":"2011","journal-title":"Drug Discov. Today"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10427","DOI":"10.1039\/c2ra20726e","article-title":"Particulate transepithelial drug carriers: Barriers and functional polymers","volume":"2","author":"Babiuch","year":"2012","journal-title":"RSC Adv."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1586\/14760584.6.3.401","article-title":"Implication of nanoparticles\/microparticles in mucosal vaccine delivery","volume":"6","author":"Vyas","year":"2007","journal-title":"Expert Rev. Vaccines"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.addr.2008.09.005","article-title":"Nanoparticles for nasal vaccination","volume":"61","author":"Csaba","year":"2009","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"797","DOI":"10.4161\/hv.27449","article-title":"Chitosan: A promising safe and immune-enhancing adjuvant for intranasal vaccines","volume":"10","author":"Smith","year":"2014","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"157","DOI":"10.2174\/157016311796799035","article-title":"Chitosan formulations as carriers for therapeutic proteins","volume":"8","author":"Andrade","year":"2011","journal-title":"Curr. Drug Discov. Technol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4812","DOI":"10.1016\/j.vaccine.2013.07.081","article-title":"In vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations","volume":"31","author":"Scherliess","year":"2013","journal-title":"Vaccine"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Ohmes, J., Saure, L.M., Sch\u00fctt, F., Trenkel, M., Seekamp, A., Scherlie\u00df, R., Adelung, R., and Fuchs, S. (2022). Injectable Thermosensitive Chitosan-Collagen Hydrogel as A Delivery System for Marine Polysaccharide Fucoidan. Mar. Drugs, 20.","DOI":"10.3390\/md20060402"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Walter, F., Winter, E., Rahn, S., Heidland, J., Meier, S., Struzek, A.-M., Lettau, M., Philipp, L.-M., Beckinger, S., and Otto, L. (2020). Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0239369"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1016\/j.ijpharm.2015.01.015","article-title":"First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice","volume":"479","author":"Scherliess","year":"2015","journal-title":"Int. J. Pharm."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2231","DOI":"10.1016\/j.vaccine.2014.01.077","article-title":"Induction of protective immunity against H1N1 influenza A(H1N1)pdm09 with spray-dried and electron-beam sterilised vaccines in non-human primates","volume":"32","author":"Scherliess","year":"2014","journal-title":"Vaccine"},{"key":"ref_25","first-page":"6077","article-title":"Design and application of chitosan microspheres as oral and nasal vaccine carriers: An updated review","volume":"7","author":"Islam","year":"2012","journal-title":"Int. J. Nanomed."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1527","DOI":"10.1166\/jbn.2016.2267","article-title":"Nanoparticle-Based Mucosal Vaccines Targeting Tumor-Associated Antigens to Human Dendritic Cells","volume":"12","author":"Baleeiro","year":"2016","journal-title":"J. Biomed. Nanotechnol."},{"key":"ref_27","unstructured":"Hanefeld, A., Weigandt, M., Wolf, M., Knolle, P., Schr\u00f6der, M., Scherlie\u00df, R., Walden, P., Diedrich, A., Steckel, H., and Baleeiro, R.B. (2014). Antigen-Loaded Chitosan Nanoparticles for Immunotherapy, Merck Patent Gesellschaft. WO2015\/185180A1."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"148","DOI":"10.3390\/vaccines3010148","article-title":"Choice and Design of Adjuvants for Parenteral and Mucosal Vaccines","volume":"3","author":"Savelkoul","year":"2015","journal-title":"Vaccines"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2562","DOI":"10.2174\/15680266113136660183","article-title":"Vaccine adjuvants: Key tools for innovative vaccine design","volume":"13","author":"Riese","year":"2013","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Brun, A. (2016). Vaccine Technologies for Veterinary Viral Diseases, Springer. Methods in Molecular Biology.","DOI":"10.1007\/978-1-4939-3008-1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.smim.2018.05.001","article-title":"Correlates of adjuvanticity: A review on adjuvants in licensed vaccines","volume":"39","author":"Rappuoli","year":"2018","journal-title":"Semin. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/978-1-4419-1132-2_13","article-title":"Immune modulators with defined molecular targets: Cornerstone to optimize rational vaccine design","volume":"655","author":"Ebensen","year":"2009","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1002\/iub.1212","article-title":"Cyclic di-nucleotide signaling enters the eukaryote domain","volume":"65","author":"Schaap","year":"2013","journal-title":"IUBMB Life"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2598","DOI":"10.1016\/j.vaccine.2010.01.013","article-title":"Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants","volume":"28","author":"Cho","year":"2010","journal-title":"Vaccine"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.imlet.2004.12.008","article-title":"From toxin to adjuvant: Basic mechanisms for the control of mucosal IgA immunity and tolerance","volume":"97","author":"Lycke","year":"2005","journal-title":"Immunol. Lett."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2534","DOI":"10.1016\/S0264-410X(00)00553-3","article-title":"Mucosal vaccines: Non toxic derivatives of LT and CT as mucosal adjuvants","volume":"19","author":"Pizza","year":"2001","journal-title":"Vaccine"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5904","DOI":"10.1128\/JVI.00296-15","article-title":"CpG Oligodeoxynucleotides Facilitate Delivery of Whole Inactivated H9N2 Influenza Virus via Transepithelial Dendrites of Dendritic Cells in Nasal Mucosa","volume":"89","author":"Qin","year":"2015","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1080\/21645515.2014.1004033","article-title":"CpG oligodeoxynucleotides as mucosal adjuvants","volume":"11","author":"Iho","year":"2015","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Nierkens, S., Brok, M.H.D., Roelofsen, T., Wagenaars, J.A.L., Figdor, C.G., Ruers, T.J., and Adema, G.J. (2009). Route of administration of the TLR9 agonist CpG critically determines the efficacy of cancer immunotherapy in mice. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0008368"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"47","DOI":"10.2174\/1874285800903010047","article-title":"The toll-like receptor 2\/6 ligand MALP-2 reduces the viability of Mycobacterium tuberculosis in murine macrophages","volume":"3","author":"Palma","year":"2009","journal-title":"Open Microbiol. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2049","DOI":"10.1016\/j.vaccine.2005.11.025","article-title":"Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime\/boost vaccination using the lipopeptide MALP-2 as adjuvant","volume":"24","author":"Borsutzky","year":"2006","journal-title":"Vaccine"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2857","DOI":"10.1002\/1521-4141(2002010)32:10<2857::AID-IMMU2857>3.0.CO;2-R","article-title":"The Mycoplasma-derived lipopeptide MALP-2 is a potent mucosal adjuvant","volume":"32","author":"Rharbaoui","year":"2002","journal-title":"Eur. J. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Skrnjug, I., Rueckert, C., Libanova, R., Lienenklaus, S., Weiss, S., and Guzm\u00e1n, C. (2014). The mucosal adjuvant cyclic di-AMP exerts immune stimulatory effects on dendritic cells and macrophages. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0095728"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Sanchez, M.V., Ebensen, T., Schulze, K., Cargnelutti, D.E., Blazejewska, P., Scodeller, E.A., and Guzm\u00e1n, C.A. (2014). Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0104824"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"5210","DOI":"10.1016\/j.vaccine.2011.05.026","article-title":"Bis-(3\u2032,5\u2032)-cyclic dimeric adenosine monophosphate: Strong Th1\/Th2\/Th17 promoting mucosal adjuvant","volume":"29","author":"Ebensen","year":"2011","journal-title":"Vaccine"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2249","DOI":"10.1016\/j.vaccine.2009.12.045","article-title":"The member of the cyclic di-nucleotide family bis-(3\u2032,5\u2032)-cyclic dimeric inosine monophosphate exerts potent activity as mucosal adjuvant","volume":"28","author":"Libanova","year":"2010","journal-title":"Vaccine"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.micinf.2011.09.002","article-title":"The overlapping host responses to bacterial cyclic dinucleotides","volume":"14","author":"Grajkowski","year":"2012","journal-title":"Microbes Infect."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3313","DOI":"10.1002\/eji.201545697","article-title":"Cyclic dinucleotides modulate human T-cell response through monocyte cell death","volume":"45","author":"Tosolini","year":"2015","journal-title":"Eur. J. Immunol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/s41541-017-0010-z","article-title":"Engineered trivalent immunogen adjuvanted with a STING agonist confers protection against Trypanosoma cruzi infection","volume":"2","author":"Bivona","year":"2017","journal-title":"NPJ Vaccines"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Matos, M.N., Cazorla, S.I., Schulze, K., Ebensen, T., Guzm\u00e1n, C.A., and Malchiodi, E.L. (2017). Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi. PLoS Negl. Trop. Dis., 11.","DOI":"10.1371\/journal.pntd.0005300"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.ebiom.2017.07.016","article-title":"Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway","volume":"22","author":"Lirussi","year":"2017","journal-title":"eBioMedicine"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"6949","DOI":"10.1016\/j.vaccine.2017.10.072","article-title":"Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes","volume":"35","author":"Landi","year":"2017","journal-title":"Vaccine"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.3389\/fimmu.2017.01223","article-title":"Mucosal Administration of Cycle-Di-Nucleotide-Adjuvanted Virosomes Efficiently Induces Protection against Influenza H5N1 in Mice","volume":"8","author":"Ebensen","year":"2017","journal-title":"Front. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Lirussi, D., Weissmann, S., Ebensen, T., Nitsche-Gloy, U., Franz, H., and Guzm\u00e1n, C. (2021). Cyclic Di-Adenosine Monophosphate: A Promising Adjuvant Candidate for the Development of Neonatal Vaccines. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13020188"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Lanfermann, C., Wintgens, S., Ebensen, T., Kohn, M., Laudeley, R., Schulze, K., Rheinheimer, C., Hegemann, J., Guzm\u00e1n, C., and Klos, A. (2021). Prophylactic Multi-Subunit Vaccine against Chlamydia trachomatis: In Vivo Evaluation in Mice. Vaccines.","DOI":"10.3390\/vaccines9060609"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"622385","DOI":"10.3389\/fimmu.2020.622385","article-title":"Self-Amplifying Pestivirus Replicon RNA Encoding Influenza Virus Nucleoprotein and Hemagglutinin Promote Humoral and Cellular Immune Responses in Pigs","volume":"11","author":"Demoulins","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"128","DOI":"10.3389\/fimmu.2020.00128","article-title":"Mucosal Heterologous Prime\/Boost Vaccination Induces Polyfunctional Systemic Immunity, Improving Protection Against Trypanosoma cruzi","volume":"11","author":"Bivona","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"4963","DOI":"10.1016\/j.vaccine.2019.07.019","article-title":"The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)\/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells","volume":"37","author":"Volckmar","year":"2019","journal-title":"Vaccine"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"31","DOI":"10.3389\/fcimb.2019.00031","article-title":"The Combination Vaccine Adjuvant System Alum\/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization","volume":"9","author":"Ebensen","year":"2019","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"7122","DOI":"10.1016\/j.vaccine.2011.05.070","article-title":"Vaccine stabilization: Research, commercialization, and potential impact","volume":"29","author":"Kristensen","year":"2011","journal-title":"Vaccine"},{"key":"ref_61","unstructured":"Lal, M. (2022, November 28). Technologies for the Development of Thermostable Vaccines. 1-24 (PATH, 2013). Available online: https:\/\/www.path.org\/media-center\/new-technology-for-producing-thermostable-vaccines\/."},{"key":"ref_62","unstructured":"Diedrich, A., and Scherlie\u00df, R. In Proceedings of the DDL 25, Edinburgh, Scotland, 10\u201312 December 2014."},{"key":"ref_63","first-page":"207","article-title":"New Horizons in the Development of Novel Needle-Free Immunization Strategies to Increase Vaccination Efficacy","volume":"398","author":"Schulze","year":"2016","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_64","first-page":"765","article-title":"Recent advances of chitosan nanoparticles as drug carriers","volume":"6","author":"Wang","year":"2011","journal-title":"Int. J. Nanomed."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"6983","DOI":"10.4049\/jimmunol.167.12.6983","article-title":"The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia","volume":"167","author":"Topham","year":"2001","journal-title":"J. Immunol."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"497364","DOI":"10.1155\/2011\/497364","article-title":"Immunogenicity of a recombinant influenza virus bearing both the CD4+ and CD8+ T cell epitopes of ovalbumin","volume":"2011","author":"Garulli","year":"2011","journal-title":"J. Biomed. Biotechnol."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/j.virol.2005.06.023","article-title":"Antigen-specific and non-specific CD4+ T cell recruitment and proliferation during influenza infection","volume":"340","author":"Chapman","year":"2005","journal-title":"Virology"},{"key":"ref_68","first-page":"11","article-title":"Flow cytometric analysis of cell division by dilution of CFSE and related dyes","volume":"9","author":"Lyons","year":"2013","journal-title":"Curr. Protoc. Cytom."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1517\/17425247.2011.565326","article-title":"Opportunities and challenges of the pulmonary route for vaccination","volume":"8","author":"Blank","year":"2011","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1111\/j.1600-065X.1997.tb00959.x","article-title":"Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo","volume":"156","author":"Pape","year":"1997","journal-title":"Immunol. Rev."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"1391","DOI":"10.1016\/j.carbpol.2011.01.045","article-title":"Chitosan oligosaccharides inhibit LPS-induced over-expression of IL-6 and TNF-\u03b1 in RAW264.7 macrophage cells through blockade of mitogen-activated protein kinase (MAPK) and PI3K\/Akt signaling pathways","volume":"84","author":"Ma","year":"2011","journal-title":"Carbohydr. Polym."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"27","DOI":"10.2174\/1567201813666160804121123","article-title":"Advances and Potential Applications of Chitosan Nanoparticles as a Delivery Carrier for the Mucosal Immunity of Vaccine","volume":"14","author":"Li","year":"2017","journal-title":"Curr. Drug Deliv."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"52691","DOI":"10.1021\/acsami.2c17627","article-title":"Enhanced Immune Responses to Mucosa by Functionalized Chitosan-Based Composite Nanoparticles as a Vaccine Adjuvant for Intranasal Delivery","volume":"14","author":"Lin","year":"2022","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"4955","DOI":"10.4049\/jimmunol.181.7.4955","article-title":"Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice","volume":"181","author":"Forbes","year":"2008","journal-title":"J. Immunol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"231","DOI":"10.4155\/tde.14.89","article-title":"Nanocarriers: A versatile approach for mucosal vaccine delivery","volume":"6","author":"Mody","year":"2015","journal-title":"Ther. Deliv."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"771","DOI":"10.3109\/1061186X.2010.523790","article-title":"Nasal vaccine innovation","volume":"18","year":"2010","journal-title":"J. Drug Target."},{"key":"ref_77","first-page":"3151","article-title":"Chitosan gel containing polymeric nanocapsules: A new formulation for vaginal drug delivery","volume":"9","author":"Frank","year":"2014","journal-title":"Int. J. Nanomed."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"1619","DOI":"10.1517\/17425247.2014.931936","article-title":"Intranasal formulations: Promising strategy to deliver vaccines","volume":"11","author":"Riese","year":"2014","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/S0169-409X(01)00197-1","article-title":"Chitosan for mucosal vaccination","volume":"52","author":"Verhoef","year":"2001","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.vetmic.2012.05.035","article-title":"Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate\/chitosan nanoparticles induces protection against highly pathogenic influenza A virus","volume":"160","author":"Moon","year":"2012","journal-title":"Vet. Microbiol."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"1051","DOI":"10.1517\/17425247.2012.697455","article-title":"Chitosan-based delivery systems for mucosal vaccines","volume":"9","author":"Watts","year":"2012","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ijpharm.2010.12.040","article-title":"Influence of particle size on regional lung deposition\u2014What evidence is there?","volume":"406","author":"Carvalho","year":"2011","journal-title":"Int. J. Pharm."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"999","DOI":"10.1007\/s11095-007-9475-1","article-title":"Pharmaceutical Particle Engineering via Spray Drying","volume":"25","author":"Vehring","year":"2008","journal-title":"Pharm. Res."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1080\/07373937.2011.620726","article-title":"Spray Drying of Mannitol as a Drug Carrier\u2014The Impact of Process Parameters on Product Properties","volume":"30","author":"Littringer","year":"2012","journal-title":"Dry. Technol."},{"key":"ref_85","unstructured":"Diedrich, A. (2015). Entwicklung einer nanopartikul\u00e4ren Formulierung zur Vakzinierung \u00fcber den Respirationstrakt. [Ph.D. Thesis, Kiel University]."},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"1909","DOI":"10.1016\/j.vaccine.2015.02.069","article-title":"The novel adjuvant dmLT promotes dose sparing, mucosal immunity and longevity of antibody responses to the inactivated polio vaccine in a murine model","volume":"33","author":"Norton","year":"2015","journal-title":"Vaccine"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1038\/mi.2008.59","article-title":"Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing","volume":"1","author":"Wee","year":"2008","journal-title":"Mucosal Immunol."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"1181","DOI":"10.1111\/irv.12056","article-title":"A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine","volume":"7","author":"Svindland","year":"2013","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1111\/j.1750-2659.2011.00271.x","article-title":"The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine","volume":"6","author":"Svindland","year":"2012","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1056\/NEJMoa030595","article-title":"Use of the inactivated intranasal influenza vaccine and the risk of Bell\u2019s palsy in Switzerland","volume":"350","author":"Mutsch","year":"2004","journal-title":"N. Engl. J. Med."},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"2169","DOI":"10.1016\/j.nano.2017.05.012","article-title":"Intranasal vaccination with an adjuvanted polyphosphazenes nanoparticle-based vaccine formulation stimulates protective immune responses in mice","volume":"13","author":"Schulze","year":"2017","journal-title":"Nanomedicine"},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"2463","DOI":"10.1016\/j.nano.2017.08.015","article-title":"Bivalent mucosal peptide vaccines administered using the LCP carrier system stimulate protective immune responses against Streptococcus pyogenes infection","volume":"13","author":"Schulze","year":"2017","journal-title":"Nanomedicine"},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.jconrel.2015.03.017","article-title":"Inverse micellar sugar glass (IMSG) nanoparticles for transfollicular vaccination","volume":"206","author":"Mittal","year":"2015","journal-title":"J. Control. Release"},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"1435","DOI":"10.1016\/j.celrep.2018.04.003","article-title":"STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection","volume":"23","author":"Sogi","year":"2018","journal-title":"Cell. Rep."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/15\/4\/1238\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:15:40Z","timestamp":1760123740000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/15\/4\/1238"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,13]]},"references-count":94,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["pharmaceutics15041238"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics15041238","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,13]]}}}