{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T20:17:03Z","timestamp":1789503423267,"version":"build-2803163510"},"reference-count":44,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T00:00:00Z","timestamp":1755561600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"GJ Buddingh Professorship, LSUHSC Foundation","award":["389090 (AJR)"],"award-info":[{"award-number":["389090 (AJR)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Effective prophylaxis for Mycobacterium tuberculosis (Mtb) requires greater understanding of immune correlates of protection. With renewed interest in BCG as an Mtb vaccine, particularly via the intravenous (IV) route, our objective was to characterize both innate and adaptive immune correlates of vaccine-induced pulmonary immunity as potential biomarkers for protective efficacy in a murine model of Mtb infection. Methods: Mice were given BCG via different routes and some boosted with recombinant virus constructs encoding Mtb Ag85B. Responding innate lymphoid cell (ILC) populations, T cells and B cells were analyzed by fluorescence activated cell sorting (FACS) for surface markers and by intracellular cytokine staining or antibody ELISPOT. Some immunized mice were challenged with aerosolized Mtb and monitored for bacterial growth in the lungs and spleen. Results: BCG given IV, but not intranasally or subcutaneously, resulted in marked increases in IFN\u03b3 expression at 72 h by pulmonary CD49+ NK cells, CD69+ ILC1, and two ILC3 populations, NCR-ILC3 and LTi cells, the latter also producing IL-22. Pulmonary ILC2 populations in these mice had significantly increased IL-13 expression at 24 h compared to the other routes. Interestingly, high levels of NK cells and ILC1 expressing IFN\u03b3 and\/or TNF\u03b1 were sustained at 8 wk, with sustained expression of IL-17A by pulmonary NCR-ILC3 and pronounced tissue-resident and effector memory CD4+ and CD8+ T cell responses. Intranasal boosting with Ad-Ag85B enhanced these T cell responses and generated Mtb-specific pulmonary IgA and IgG B cells, correlating with significantly reduced bacterial loads following Mtb challenge. Conclusions: BCG given IV primed for both early and persistent pulmonary ILC1\/ILC3 responses of a predominantly Th1\/Th17-type profile along with local Mtb-specific memory T cell and B cell populations, correlating with enhanced protective efficacy. These are worthy of further study as compartmentalized biomarkers for effective vaccine-induced local immunity against Mtb.<\/jats:p>","DOI":"10.3390\/vaccines13080876","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T11:41:41Z","timestamp":1755603701000},"page":"876","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["BCG Vaccine-Induced Innate and Adaptive Pulmonary Immunity Correlating with Protective Efficacy Against Mycobacterium tuberculosis in the Lungs"],"prefix":"10.3390","volume":"13","author":[{"given":"Mayank","family":"Khanna","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, and the Louisiana Vaccine Center, New Orleans, LA 70112, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3506-4544","authenticated-orcid":false,"given":"Alistair J.","family":"Ramsay","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, and the Louisiana Vaccine Center, New Orleans, LA 70112, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,19]]},"reference":[{"key":"ref_1","unstructured":"WHO (2024). Global Tuberculosis Report."},{"key":"ref_2","first-page":"59","article-title":"Expanded programme on immunization","volume":"41","author":"Keja","year":"1988","journal-title":"World Health Stat. Q."},{"key":"ref_3","first-page":"698","article-title":"Efficacy of BCG vaccine in the prevention of tuberculosis","volume":"271","author":"Colditz","year":"1994","journal-title":"Meta-Anal. Publ. Lit. JAMA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1016\/S0140-6736(13)60177-4","article-title":"Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: A randomised, placebo-controlled phase 2b trial","volume":"381","author":"Tameris","year":"2013","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1832","DOI":"10.1093\/infdis\/jir195","article-title":"Dose-finding study of the novel tuberculosis vaccine, MVA85A, in healthy BCG-vaccinated infants","volume":"203","author":"Scriba","year":"2011","journal-title":"J. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e87340","DOI":"10.1371\/journal.pone.0087340","article-title":"The Candidate TB Vaccine, MVA85A, Induces Highly Durable Th1 Responses","volume":"9","author":"Tameris","year":"2014","journal-title":"PLoS ONE"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2429","DOI":"10.1056\/NEJMoa1909953","article-title":"Final Analysis of a Trial of M72\/AS01(E) Vaccine to Prevent Tuberculosis","volume":"381","author":"Tait","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1038\/s41586-019-1817-8","article-title":"Prevention of tuberculosis in macaques after intravenous BCG immunization","volume":"577","author":"Darrah","year":"2020","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"640916","DOI":"10.3389\/fimmu.2021.640916","article-title":"Local Pulmonary Immunological Biomarkers in Tuberculosis","volume":"12","author":"Morrison","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41591-018-0319-9","article-title":"Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques","volume":"25","author":"Dijkman","year":"2019","journal-title":"Nat. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1016\/j.chom.2023.05.006","article-title":"Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-Mediat","volume":"31","author":"Darrah","year":"2023","journal-title":"Prot. Against Tuberc. Rhesus Macaques. Cell Host Microbe"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"111128","DOI":"10.1016\/j.isci.2024.111128","article-title":"Humoral correlates of protection against Mycobacterium tuberculosis following intravenous BCG vaccination in rhesus macaques","volume":"27","author":"Irvine","year":"2024","journal-title":"iScience"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"eadq8229","DOI":"10.1126\/sciadv.adq8229","article-title":"High-dose intravenous BCG vaccination induces enhanced immune signaling in the airways","volume":"11","author":"Peters","year":"2025","journal-title":"Sci. Adv."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.coi.2015.01.004","article-title":"Innate lymphoid cells: New insights into function and development","volume":"32","author":"Cortez","year":"2015","journal-title":"Curr. Opin. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1126\/science.aac9593","article-title":"Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs","volume":"350","author":"Gasteiger","year":"2015","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.immuni.2009.06.008","article-title":"Interleukin-22-producing natural killer cells and lymphoid tissue inducer-like cells in mucosal immunity","volume":"31","author":"Colonna","year":"2009","journal-title":"Immunity"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"aaa6566","DOI":"10.1126\/science.aaa6566","article-title":"Innate lymphoid cells. Innate lymphoid cells: A new paradigm in immunology","volume":"348","author":"Eberl","year":"2015","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2768","DOI":"10.1016\/j.vaccine.2012.02.020","article-title":"CD40 ligand enhances immunogenicity of vector-based vaccines in immunocompetent and CD4+ T cell deficient individuals","volume":"30","author":"Auten","year":"2012","journal-title":"Vaccine"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1780","DOI":"10.1016\/j.vaccine.2021.01.071","article-title":"Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates","volume":"39","author":"Khanna","year":"2021","journal-title":"Vaccine"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e0136635","DOI":"10.1371\/journal.pone.0136635","article-title":"DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection","volume":"10","author":"Dalmia","year":"2015","journal-title":"PLoS ONE"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e0148701","DOI":"10.1371\/journal.pone.0148701","article-title":"Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant","volume":"11","author":"Rady","year":"2016","journal-title":"PLoS ONE"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1198310","DOI":"10.3389\/fimmu.2023.1198310","article-title":"Flow cytometric analysis of innate lymphoid cells: Challenges and solutions","volume":"14","author":"Sadeghalvad","year":"2023","journal-title":"Front. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1164\/rccm.201006-0963PP","article-title":"Human lung immunity against Mycobacterium tuberculosis: Insights into pathogenesis and protection","volume":"183","author":"Schwander","year":"2011","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1136\/thoraxjnl-2014-205202","article-title":"TB Vaccine Development: Where Are We Why Is. It So Difficult?","volume":"70","author":"Wilkie","year":"2015","journal-title":"Thorax"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4634","DOI":"10.1128\/IAI.00517-06","article-title":"Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis","volume":"74","author":"Santosuosso","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/j.ijid.2014.11.026","article-title":"Tuberculosis vaccines--state of the art, and novel approaches to vaccine development","volume":"32","author":"Walker","year":"2015","journal-title":"Int. J. Infect. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"582414","DOI":"10.3389\/fimmu.2020.582414","article-title":"Antigens of Mycobacterium tuberculosis Stimulate CXCR6+ Natural Killer Cells","volume":"11","author":"Zuniga","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1038\/s41590-023-01476-3","article-title":"Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control","volume":"24","author":"Swanson","year":"2023","journal-title":"Nat. Immunol."},{"key":"ref_29","first-page":"2565","article-title":"Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increased susceptibility to intranasal infection with Mycobacterium bovis BCG","volume":"23","author":"Rodriguez","year":"2005","journal-title":"Vaccine"},{"key":"ref_30","first-page":"706","article-title":"An IgG antibody response to the antigen 85 complex is associated with good outcome in Mexican Totonaca Indians with pulmonary tuberculosis","volume":"6","author":"Mancilla","year":"2002","journal-title":"Int. J. Tuberc. Lung D"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1016\/j.cell.2016.08.072","article-title":"A Functional Role for Antibodies in Tuberculosis","volume":"167","author":"Lu","year":"2016","journal-title":"Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1038\/s41577-018-0028-0","article-title":"Antibodies and Tuberculosis: Finally Coming Age?","volume":"18","author":"Li","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"10-1128","DOI":"10.1128\/mBio.01686-16","article-title":"Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis","volume":"7","author":"Perdomo","year":"2016","journal-title":"mBio"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1038\/s41385-018-0109-1","article-title":"Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1(+) KLRG1(\u2212) CD4(+) T cells","volume":"12","author":"Bull","year":"2019","journal-title":"Mucosal Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1038\/ni.3484","article-title":"Innate lymphoid cell function in the context of adaptive immunity","volume":"17","author":"Bando","year":"2016","journal-title":"Nat. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1006\/cimm.1994.1196","article-title":"Interleukin-12 (IL-12) augments cytolytic activity of natural killer cells toward Mycobacterium tuberculosis-infected human monocytes","volume":"156","author":"Denis","year":"1994","journal-title":"Cell Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1164\/ajrccm.158.2.9707102","article-title":"CD4(+) T cell and natural killer cell-dependent killing of Mycobacterium tuberculosis by human monocytes","volume":"158","author":"Yoneda","year":"1998","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"130","DOI":"10.4049\/jimmunol.172.1.130","article-title":"NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen","volume":"172","author":"Vankayalapati","year":"2004","journal-title":"J. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"7151","DOI":"10.4049\/jimmunol.151.12.7151","article-title":"Modulation of murine macrophage function by IL-13","volume":"151","author":"Doherty","year":"1993","journal-title":"J. Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1146\/annurev-immunol-032712-095906","article-title":"Interleukin-4- and interleukin-13-mediated alternatively activated macrophages: Roles in homeostasis and disease","volume":"31","author":"Locksley","year":"2013","journal-title":"Annu. Rev. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1038\/ni1449","article-title":"IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge","volume":"8","author":"Khader","year":"2007","journal-title":"Nat. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3786","DOI":"10.4049\/jimmunol.178.6.3786","article-title":"IL-17-mediated regulation of innate and acquired immune response against pulmonary bacille Calmette-Guerin infection","volume":"178","author":"Umemura","year":"2007","journal-title":"J. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1038\/s41586-019-1276-2","article-title":"Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis","volume":"570","author":"Ardain","year":"2019","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1111\/imcb.12007","article-title":"BCG vaccination drives accumulation and effector function of innate lymphoid cells in murine lungs","volume":"96","author":"Steigler","year":"2018","journal-title":"Immunol. Cell Biol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/8\/876\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:30:41Z","timestamp":1760034641000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/8\/876"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,19]]},"references-count":44,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["vaccines13080876"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13080876","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,19]]}}}