{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T05:38:39Z","timestamp":1762321119124,"version":"build-2065373602"},"reference-count":42,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T00:00:00Z","timestamp":1762128000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["1R43AI165089"],"award-info":[{"award-number":["1R43AI165089"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: Intranasal (I.N.) vaccination holds promise to elicit mucosal immunity that counters respiratory pathogens at the site of infection. For subunit protein vaccines, immunostimulatory adjuvants are typically required. Methods: We screened a panel of 22 lipid-phase adjuvants to identify which ones elicited antigen-specific IgA with I.N. immunization of liposome-displayed SARS-CoV-2 receptor-binding domain (RBD). Results: Initial screening showed the TLR-4 agonist Kdo2-Lipid A (KLA) effectively elicited RBD-specific IgA. A second round of screening identified further inclusion of the invariant NKT cell ligands \u03b1-Galactosylceramide (\u03b1-GalCer) and its synthetic analog 7DW8-5 as complementary adjuvants for I.N. immunization, resulting in orders-of-magnitude-greater mucosal IgA response relative to intramuscular (I.M.) immunization. The inclusion of cationic lipids conferred capacity for mucosal adhesion and maintained immune responses. In K18 hACE2 transgenic mice, vaccination significantly reduced viral replication and prevented mortality from SARS-CoV-2 challenge. Conclusions: These results point towards the potential for the use of KLA and \u03b1-GalCer for I.N. subunit vaccines.<\/jats:p>","DOI":"10.3390\/pharmaceutics17111422","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:47:58Z","timestamp":1762177678000},"page":"1422","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["In Vivo Iterative Adjuvant Screening Identifies an Intranasal Vaccine Formulation for Elicitation of Protective Mucosal Immune Responses Against SARS-CoV-2"],"prefix":"10.3390","volume":"17","author":[{"given":"Yang","family":"Jiao","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1011-5608","authenticated-orcid":false,"given":"Sara H.","family":"Mahmoud","sequence":"additional","affiliation":[{"name":"Host-Pathogen Interactions (HPI) and Disease Intervention and Prevention (DIP) Programs, Texas Biomedical Research Institute, San Antonio, TX 78227, USA"},{"name":"Center of Scientific Excellence for Influenza Viruses, National Research Centre, Giza 12622, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chengjin","family":"Ye","sequence":"additional","affiliation":[{"name":"Host-Pathogen Interactions (HPI) and Disease Intervention and Prevention (DIP) Programs, Texas Biomedical Research Institute, San Antonio, TX 78227, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuan","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei-Chiao","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qinzhe","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0530-9512","authenticated-orcid":false,"given":"Shiqi","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiting","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8587-277X","authenticated-orcid":false,"given":"Moriya","family":"Tsuji","sequence":"additional","affiliation":[{"name":"Aaron Diamond AIDS Research Center, Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7084-0804","authenticated-orcid":false,"given":"Luis","family":"Martinez-Sobrido","sequence":"additional","affiliation":[{"name":"Host-Pathogen Interactions (HPI) and Disease Intervention and Prevention (DIP) Programs, Texas Biomedical Research Institute, San Antonio, TX 78227, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9052-884X","authenticated-orcid":false,"given":"Jonathan F.","family":"Lovell","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 14260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chakraborty, C., Bhattacharya, M., and Dhama, K. (2023). SARS-CoV-2 Vaccines, Vaccine Development Technologies, and Significant Efforts in Vaccine Development during the Pandemic: The Lessons Learned Might Help to Fight against the Next Pandemic. Vaccines, 11.","DOI":"10.3390\/vaccines11030682"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1038\/s41586-020-2798-3","article-title":"SARS-CoV-2 vaccines in development","volume":"586","author":"Krammer","year":"2020","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2619","DOI":"10.1016\/j.drudis.2021.07.021","article-title":"Intranasal vaccines for SARS-CoV-2: From challenges to potential in COVID-19 management","volume":"26","author":"Chavda","year":"2021","journal-title":"Drug Discov. Today"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Tokunoh, N., Tamiya, S., Watanabe, M., Okamoto, T., Anindita, J., Tanaka, H., Ono, C., Hirai, T., Akita, H., and Matsuura, Y. (2023). A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1224634"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1038\/nri3251","article-title":"Recent progress in mucosal vaccine development: Potential and limitations","volume":"12","author":"Lycke","year":"2012","journal-title":"Nat. Rev. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1007\/s13346-012-0085-z","article-title":"Strategies for intranasal delivery of vaccines","volume":"3","author":"Zaman","year":"2013","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1080\/21645515.2015.1114195","article-title":"Induction of mucosal immunity through systemic immunization: Phantom or reality?","volume":"12","author":"Su","year":"2016","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"eadh1655","DOI":"10.1126\/sciadv.adh1655","article-title":"Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection","volume":"9","author":"Meyer","year":"2023","journal-title":"Sci. Adv."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"16757","DOI":"10.1021\/acsnano.2c06350","article-title":"Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation COVID-19 Vaccine","volume":"16","author":"Lam","year":"2022","journal-title":"ACS Nano"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e12192","DOI":"10.1002\/jev2.12192","article-title":"A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants","volume":"11","author":"Jiang","year":"2022","journal-title":"J. Extracell. Vesicles"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1038\/s41541-023-00717-8","article-title":"Phase III Pivotal comparative clinical trial of intranasal (iNCOVACC) and intramuscular COVID-19 vaccine (Covaxin\u00ae)","volume":"8","author":"Singh","year":"2023","journal-title":"npj Vaccines"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"102374","DOI":"10.1016\/j.eclinm.2023.102374","article-title":"Short-term effectiveness of single-dose intranasal spray COVID-19 vaccine against symptomatic SARS-CoV-2 Omicron infection in healthcare workers: A prospective cohort study","volume":"67","author":"Mi","year":"2024","journal-title":"eClinicalMedicine"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1038\/s41541-025-01096-y","article-title":"A randomized phase I trial of intranasal SARS-CoV-2 vaccine dNS1-RBD in children aged 3\u201317 years","volume":"10","author":"Chu","year":"2025","journal-title":"npj Vaccines"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Worzner, K., Schmidt, S.T., Zimmermann, J., Tami, A., Polacek, C., Fernandez-Antunez, C., Hartmann, K.T., Jensen, R.F., Hansen, J.S., and Illigen, K. (2025). Intranasal recombinant protein subunit vaccine targeting TLR3 induces respiratory tract IgA and CD8 T cell responses and protects against respiratory virus infection. eBioMedicine, 113.","DOI":"10.1016\/j.ebiom.2025.105615"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1038\/s41541-022-00451-7","article-title":"Intranasal administration of BReC-CoV-2 COVID-19 vaccine protects K18-hACE2 mice against lethal SARS-CoV-2 challenge","volume":"7","author":"Wong","year":"2022","journal-title":"NPJ Vaccines"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Frank, N., Dickinson, D., Lovett, G., Liu, Y., Yu, H., Cai, J., Yao, B., Jiang, X., and Hsu, S. (2024). Evaluation of Novel Nasal Mucoadhesive Nanoformulations Containing Lipid-Soluble EGCG for Long COVID Treatment. Pharmaceutics, 16.","DOI":"10.20944\/preprints202404.1974.v1"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1080\/14760584.2023.2274479","article-title":"Vaccine approaches for antigen capture by liposomes","volume":"22","author":"Zhou","year":"2023","journal-title":"Expert Rev. Vaccines"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1177\/2051013614541440","article-title":"Liposomes as vaccine delivery systems: A review of the recent advances","volume":"2","author":"Schwendener","year":"2014","journal-title":"Ther. Adv. Vaccines"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Rehman, M., Tahir, N., Sohail, M.F., Qadri, M.U., Duarte, S.O.D., Brandao, P., Esteves, T., Javed, I., and Fonte, P. (2024). Lipid-Based Nanoformulations for Drug Delivery: An Ongoing Perspective. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16111376"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Huang, W.C., Mabrouk, M.T., Zhou, L., Baba, M., Tachibana, M., Torii, M., Takashima, E., Locke, E., Plieskatt, J., and King, C.R. (2022). Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages. Commun. Biol., 5.","DOI":"10.1038\/s42003-022-03688-z"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Huang, W.C., Chiem, K., Martinez-Sobrido, L., and Lovell, J.F. (2022). Intranasal Immunization with Liposome-Displayed Receptor-Binding Domain Induces Mucosal Immunity and Protection against SARS-CoV-2. Pathogens, 11.","DOI":"10.3390\/pathogens11091035"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2005637","DOI":"10.1002\/adma.202005637","article-title":"SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination","volume":"32","author":"Huang","year":"2020","journal-title":"Adv. Mater."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e2304534","DOI":"10.1002\/smll.202304534","article-title":"SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ from Lyophilized Lipids","volume":"20","author":"Jiao","year":"2024","journal-title":"Small"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e29927","DOI":"10.1002\/jmv.29927","article-title":"Interim safety and immunogenicity analysis of the EuCorVac-19 COVID-19 vaccine in a Phase 3 randomized, observer-blind, immunobridging trial in the Philippines","volume":"96","author":"Lovell","year":"2024","journal-title":"J. Med. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Lovell, J.F., Miura, K., Baik, Y.O., Lee, C., Choi, Y., Lee, J.Y., Long, C.A., Ylade, M., Lee-Llacer, R., and De Asis, N. (2025). Year-Long Antibody Response to the EuCorVac-19 SARS-CoV-2 Vaccine in Healthy Filipinos. Vaccines, 13.","DOI":"10.3390\/vaccines13080776"},{"key":"ref_26","first-page":"189","article-title":"Iterative selection of lipid nanoparticle vaccine adjuvants for rapid elicitation of tumoricidal CD8+ T cells","volume":"48","author":"Luo","year":"2025","journal-title":"Bioact. Mater."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1111\/brv.12114","article-title":"Kdo2-lipid A: Structural diversity and impact on immunopharmacology","volume":"90","author":"Wang","year":"2015","journal-title":"Biol. Rev. Camb. Philos. Soc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"38568","DOI":"10.1074\/jbc.M110.170621","article-title":"Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy","volume":"285","author":"Sims","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2328","DOI":"10.1172\/JCI26297","article-title":"Activation or anergy: NKT cells are stunned by alpha-galactosylceramide","volume":"115","author":"Sullivan","year":"2005","journal-title":"J. Clin. Investig."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e20240728","DOI":"10.1084\/jem.20240728","article-title":"Identification of \u03b1-galactosylceramide as an endogenous mammalian antigen for iNKT cells","volume":"222","author":"Hosono","year":"2025","journal-title":"J. Exp. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1038\/ni827","article-title":"Prolonged IFN-gamma-producing NKT response induced with alpha-galactosylceramide-loaded DCs","volume":"3","author":"Fujii","year":"2002","journal-title":"Nat. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"12921","DOI":"10.3389\/jpps.2024.12921","article-title":"Chitosan non-particulate vaccine delivery systems","volume":"27","author":"Masimov","year":"2024","journal-title":"J. Pharm. Pharm. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e09394","DOI":"10.1016\/j.heliyon.2022.e09394","article-title":"Liposomes: Structure, composition, types, and clinical applications","volume":"8","author":"Nsairat","year":"2022","journal-title":"Heliyon"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Shawky, S., Makled, S., Awaad, A., and Boraie, N. (2022). Quercetin Loaded Cationic Solid Lipid Nanoparticles in a Mucoadhesive In Situ Gel-A Novel Intravesical Therapy Tackling Bladder Cancer. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14112527"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2307","DOI":"10.1039\/c1nr10166h","article-title":"The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses","volume":"3","author":"Ma","year":"2011","journal-title":"Nanoscale"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"113995","DOI":"10.1016\/j.jviromet.2020.113995","article-title":"Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2","volume":"287","author":"Park","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Feng, H., Sun, R., Song, G., Zhu, S., Nie, Z., Lin, L., Yi, R., Wu, S., Wang, G., and He, Y. (2022). A Glycolipid \u03b1-GalCer Derivative, 7DW8-5 as a Novel Mucosal Adjuvant for the Split Inactivated Influenza Vaccine. Viruses, 14.","DOI":"10.3390\/v14061174"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Padte, N.N., Boente-Carrera, M., Andrews, C.D., McManus, J., Grasperge, B.F., Gettie, A., Coelho-dos-Reis, J.G., Li, X., Wu, D., and Bruder, J.T. (2013). A glycolipid adjuvant, 7DW8-5, enhances CD8+ T cell responses induced by an adenovirus-vectored malaria vaccine in non-human primates. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0078407"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/s41541-023-00691-1","article-title":"Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice","volume":"8","author":"Jangra","year":"2023","journal-title":"NPJ Vaccines"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Kawai, A., Tokunoh, N., Kawahara, E., Tamiya, S., Okamura, S., Ono, C., Anindita, J., Tanaka, H., Akita, H., and Yamasaki, S. (2023). Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses. J. Clin. Investig., 133.","DOI":"10.1172\/JCI166827"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1540","DOI":"10.4049\/jimmunol.175.3.1540","article-title":"Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells","volume":"175","author":"Biburger","year":"2005","journal-title":"J. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Artiaga, B.L., Madden, D., Kwon, T., McDowell, C., Keating, C., Balaraman, V., de Carvahlo Madrid, D.M., Touchard, L., Henningson, J., and Meade, P. (2024). Adjuvant Use of the Invariant-Natural-Killer-T-Cell Agonist alpha-Galactosylceramide Leads to Vaccine-Associated Enhanced Respiratory Disease in Influenza-Vaccinated Pigs. Vaccines, 12.","DOI":"10.3390\/vaccines12091068"}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/11\/1422\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T05:24:38Z","timestamp":1762320278000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/11\/1422"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,3]]},"references-count":42,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["pharmaceutics17111422"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics17111422","relation":{},"ISSN":["1999-4923"],"issn-type":[{"type":"electronic","value":"1999-4923"}],"subject":[],"published":{"date-parts":[[2025,11,3]]}}}