{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T16:55:06Z","timestamp":1791219306323,"version":"4.1.0"},"reference-count":51,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2017,12,6]],"date-time":"2017-12-06T00:00:00Z","timestamp":1512518400000},"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":"publisher","award":["AI96967"],"award-info":[{"award-number":["AI96967"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI036211"],"award-info":[{"award-number":["AI036211"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Infection by Mycobacterium tuberculosis (Mtb) remains a major global concern and the available Bacillus Calmette-Guerin (BCG) vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (\u03b1-GalCer), a potent natural killer T (NKT) cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the \u03b1-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in na\u00efve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-\u03b3 and IL-2 producing T cells.<\/jats:p>","DOI":"10.3390\/vaccines5040047","type":"journal-article","created":{"date-parts":[[2017,12,6]],"date-time":"2017-12-06T11:29:36Z","timestamp":1512559776000},"page":"47","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice"],"prefix":"10.3390","volume":"5","author":[{"given":"Arshad","family":"Khan","sequence":"first","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, The University of Texas Health Science Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shailbala","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8398-7477","authenticated-orcid":false,"given":"Gloria","family":"Galvan","sequence":"additional","affiliation":[{"name":"Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA"},{"name":"UTHealth Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chinnaswamy","family":"Jagannath","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, The University of Texas Health Science Center, Houston, TX 77030, USA"},{"name":"UTHealth Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K.","family":"Sastry","sequence":"additional","affiliation":[{"name":"Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA"},{"name":"UTHealth Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA"},{"name":"Department of Veterinary Sciences, The University of Texas M.D. Anderson Cancer Center, Bastrop, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,12,6]]},"reference":[{"key":"ref_1","unstructured":"WHO (2017, October 01). Global Tuberculosis Report 2017. Available online: http:\/\/www.who.int\/tb\/publications\/global_report\/en\/."},{"key":"ref_2","unstructured":"Adigun, R., and Bhimji, S.S. (2017). Tuberculosis. Statpearls, StatPearls Publishing LLC."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2733","DOI":"10.1111\/ajt.14471","article-title":"Solid organ transplant-transmitted tuberculosis linked to a community outbreak\u2014California, 2015","volume":"17","author":"Kay","year":"2017","journal-title":"Am. J. Transplant."},{"key":"ref_4","first-page":"267","article-title":"Risk factors associated with tuberculosis mortality in adults in six provinces of argentina","volume":"77","author":"Zerbini","year":"2017","journal-title":"Medicina"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e10","DOI":"10.1016\/S2214-109X(14)70361-4","article-title":"The who 2014 global tuberculosis report\u2014Further to go","volume":"3","author":"Zumla","year":"2015","journal-title":"Lancet Glob. Health"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1128\/CMR.00042-10","article-title":"Hiv and tuberculosis: A deadly human syndemic","volume":"24","author":"Kwan","year":"2011","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Tavares, A.M., Fronteira, I., Couto, I., Machado, D., Viveiros, M., Abecasis, A.B., and Dias, S. (2017). HIV and tuberculosis co-infection among migrants in europe: A systematic review on the prevalence, incidence and mortality. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0185526"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"S64","DOI":"10.1086\/314072","article-title":"Preventing tuberculosis with bacillus calmette-guerin vaccine: A meta-analysis of the literature","volume":"31","author":"Brewer","year":"2000","journal-title":"Clin. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.1080\/14712598.2017.1378641","article-title":"Successes and failures in human tuberculosis vaccine development","volume":"17","year":"2017","journal-title":"Expert Opin. Biol. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1086\/499953","article-title":"Bacille Calmette-Gu\u00e9rin vaccine-induced disease in HIV-infected and HIV-uninfected children","volume":"42","author":"Hesseling","year":"2006","journal-title":"Clin. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6148","DOI":"10.1128\/IAI.72.10.6148-6150.2004","article-title":"Protective effect of a tuberculosis subunit vaccine based on a fusion of antigen 85b and esat-6 in the aerosol guinea pig model","volume":"72","author":"Olsen","year":"2004","journal-title":"Infect. Immun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2773","DOI":"10.1128\/IAI.69.5.2773-2778.2001","article-title":"Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6","volume":"69","author":"Andersen","year":"2001","journal-title":"Infect. Immun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3504","DOI":"10.4049\/jimmunol.162.6.3504","article-title":"Structural deficiencies in granuloma formation in tnf gene-targeted mice underlie the heightened susceptibility to aerosol mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin","volume":"162","author":"Bean","year":"1999","journal-title":"J. Immun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2249","DOI":"10.1084\/jem.178.6.2249","article-title":"An essential role for interferon gamma in resistance to mycobacterium tuberculosis infection","volume":"178","author":"Flynn","year":"1993","journal-title":"J. Exp. Med."},{"key":"ref_15","first-page":"1","article-title":"Mucosal delivery routes for optimal immunization: Targeting immunity to the right tissues","volume":"354","author":"Czerkinsky","year":"2012","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1615\/CritRevTherDrugCarrierSyst.2014010102","article-title":"Mucosal immunization: A review of strategies and challenges","volume":"31","author":"Patel","year":"2014","journal-title":"Crit. Rev. Ther. Drug Carr. Syst."},{"key":"ref_17","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_18","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_19","doi-asserted-by":"crossref","unstructured":"Singh, S., Yang, G., Schluns, K.S., Anthony, S.M., and Sastry, K.J. (2014). Sublingual vaccination induces mucosal and systemic adaptive immunity for protection against lung tumor challenge. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0090001"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/j.coi.2012.03.014","article-title":"Vaccines against mucosal infections","volume":"24","author":"Holmgren","year":"2012","journal-title":"Curr. Opin. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"11252","DOI":"10.1073\/pnas.0604812103","article-title":"Glycolipid alpha-c-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice","volume":"103","author":"Fujii","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1007\/s00262-007-0373-5","article-title":"Phase i study of alpha-galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer","volume":"57","author":"Uchida","year":"2008","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Lu, G., Zhou, A., Meng, M., Wang, L., Han, Y., Guo, J., Zhou, H., Cong, H., Zhao, Q., and Zhu, X.Q. (2014). Alpha-galactosylceramide enhances protective immunity induced by DNA vaccine of the sag5d gene of toxoplasma gondii. BMC Infect. Dis., 14.","DOI":"10.1186\/s12879-014-0706-x"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3312","DOI":"10.1002\/eji.201041359","article-title":"Intranasal but not intravenous delivery of the adjuvant alpha-galactosylceramide permits repeated stimulation of natural killer T cells in the lung","volume":"41","author":"Courtney","year":"2011","journal-title":"Eur. J. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"241","DOI":"10.2174\/0929867043456115","article-title":"Alpha-galactosylceramide: Potential immunomodulatory activity and future application","volume":"11","author":"Hayakawa","year":"2004","journal-title":"Curr. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"6435","DOI":"10.4049\/jimmunol.0900136","article-title":"The mucosal adjuvant effect of alpha-galactosylceramide for induction of protective immunity to sexually transmitted viral infection","volume":"182","author":"Lindqvist","year":"2009","journal-title":"J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3335","DOI":"10.1016\/j.vaccine.2009.01.083","article-title":"Alpha-galactosylceramide is an effective mucosal adjuvant for repeated intranasal or oral delivery of hiv peptide antigens","volume":"27","author":"Courtney","year":"2009","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"8598","DOI":"10.1016\/j.vaccine.2007.09.073","article-title":"Sublingual immunization induces broad-based systemic and mucosal immune responses in mice","volume":"25","author":"Cuburu","year":"2007","journal-title":"Vaccine"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"686","DOI":"10.3390\/vaccines2040686","article-title":"Enhancement of mucosal immunogenicity of viral vectored vaccines by the nkt cell agonist alpha-galactosylceramide as adjuvant","volume":"2","author":"Singh","year":"2014","journal-title":"Vaccines"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1128\/AAC.47.2.783-785.2003","article-title":"Rapid in vivo screening of experimental drugs for tuberculosis using gamma interferon gene-disrupted mice","volume":"47","author":"Lenaerts","year":"2003","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1128\/IAI.73.1.546-551.2005","article-title":"Replication dynamics of mycobacterium tuberculosis in chronically infected mice","volume":"73","author":"Timm","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1093\/intimm\/12.7.987","article-title":"The interface between innate and acquired immunity: Glycolipid antigen presentation by cd1d-expressing dendritic cells to nkt cells induces the differentiation of antigen-specific cytotoxic T lymphocytes","volume":"12","author":"Nishimura","year":"2000","journal-title":"Int. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3271","DOI":"10.4049\/jimmunol.161.7.3271","article-title":"Selective ability of mouse cd1 to present glycolipids: Alpha-galactosylceramide specifically stimulates v alpha 14+ nk T lymphocytes","volume":"161","author":"Burdin","year":"1998","journal-title":"J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1164\/rccm.200912-1921OC","article-title":"Alpha-galactosylceramide as a therapeutic agent for pulmonary mycobacterium tuberculosis infection","volume":"182","author":"Skold","year":"2010","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"24508","DOI":"10.1074\/jbc.M111.234062","article-title":"The Mycobacterium tuberculosis Early Secreted Antigenic Target of 6 kDa Inhibits T Cell Interferon-\u03b3 Production through the p38 Mitogen-activated Protein Kinase Pathway","volume":"286","author":"Peng","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Jang, A.-R., Choi, J.-H., Shin, S.J., and Park, J.-H. (2017). Mycobacterium tuberculosis ESAT6 induces IFN-\u03b2 gene expression in Macrophages via TLRs-mediated signaling. Cytokine.","DOI":"10.1016\/j.cyto.2017.10.006"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3143","DOI":"10.4049\/jimmunol.178.5.3143","article-title":"The Type I IFN Response to Infection with Mycobacterium tuberculosis Requires ESX-1-Mediated Secretion and Contributes to Pathogenesis","volume":"178","author":"Stanley","year":"2007","journal-title":"J. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1121","DOI":"10.1084\/jem.189.7.1121","article-title":"The natural killer T (nkt) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (il)-12 production by dendritic cells and il-12 receptor expression on nkt cells","volume":"189","author":"Kitamura","year":"1999","journal-title":"J. Exp. Med."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2373","DOI":"10.4049\/jimmunol.163.5.2373","article-title":"Cutting edge: Activation of nk T cells by cd1d and alpha-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype","volume":"163","author":"Singh","year":"1999","journal-title":"J. Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1038\/nri854","article-title":"The unconventional lifestyle of nkt cells","volume":"2","author":"Kronenberg","year":"2002","journal-title":"Nat. Rev. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"6302","DOI":"10.1128\/IAI.70.11.6302-6309.2002","article-title":"Activation of nkt cells protects mice from tuberculosis","volume":"70","author":"Chackerian","year":"2002","journal-title":"Infect. Immun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2100","DOI":"10.1128\/IAI.06018-11","article-title":"Dysfunction of natural killer T cells in patients with active mycobacterium tuberculosis infection","volume":"80","author":"Kee","year":"2012","journal-title":"Infect. Immun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2176","DOI":"10.1021\/jm00012a018","article-title":"Structure-activity relationship ofAlpha-galactosylceramides against b16-bearing mice","volume":"38","author":"Morita","year":"1995","journal-title":"J. Med. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1084\/jem.192.7.921","article-title":"Natural killer T cell activation inhibits hepatitis b virus replication in vivo","volume":"192","author":"Kakimi","year":"2000","journal-title":"J. Exp. Med."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1084\/jem.20011889","article-title":"Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines","volume":"195","author":"Bergmann","year":"2002","journal-title":"J. Exp. Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1644","DOI":"10.1073\/pnas.0708684105","article-title":"Sublingual vaccination with influenza virus protects mice against lethal viral infection","volume":"105","author":"Song","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1038\/mi.2013.59","article-title":"Role of type 1 natural killer T cells in pulmonary immunity","volume":"6","author":"Paget","year":"2013","journal-title":"Mucosal Immunol."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Rothchild, A.C., Jayaraman, P., Nunes-Alves, C., and Behar, S.M. (2014). iNKT cell production of GM-CSF controls Mycobacterium tuberculosis. PLoS Pathog., 10.","DOI":"10.1371\/journal.ppat.1003805"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1038\/mi.2009.99","article-title":"Nkt cells in mucosal immunity","volume":"2","author":"Middendorp","year":"2009","journal-title":"Mucosal Immunol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"4305","DOI":"10.4049\/jimmunol.165.8.4305","article-title":"Selective bystander proliferation of memory cd4+ and cd8+ T cells upon nk T or T cell activation","volume":"165","author":"Eberl","year":"2000","journal-title":"J. Immunol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.tube.2010.06.002","article-title":"The achilles heel of bcg","volume":"90","author":"Orme","year":"2010","journal-title":"Tuberculosis"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/5\/4\/47\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:52:48Z","timestamp":1760208768000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/5\/4\/47"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,12,6]]},"references-count":51,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2017,12]]}},"alternative-id":["vaccines5040047"],"URL":"https:\/\/doi.org\/10.3390\/vaccines5040047","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,12,6]]}}}