{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T16:49:03Z","timestamp":1778863743652,"version":"3.51.4"},"reference-count":44,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2022,6,10]],"date-time":"2022-06-10T00:00:00Z","timestamp":1654819200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hebei Province University Science and Technology Research Key Project","award":["ZD2016030"],"award-info":[{"award-number":["ZD2016030"]}]},{"DOI":"10.13039\/501100003482","name":"Department of Education of Hebei Province","doi-asserted-by":"crossref","award":["QN2015024"],"award-info":[{"award-number":["QN2015024"]}],"id":[{"id":"10.13039\/501100003482","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Nontypeable <jats:italic>Haemophilus influenzae<\/jats:italic> (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH<jats:sub>3<\/jats:sub>CN), and P6-MCMs with an average size of 590.4\u00b116.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1\/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4<jats:sup>+<\/jats:sup>-mediated Th immune responses and CD8<jats:sup>+<\/jats:sup>-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.<\/jats:p>","DOI":"10.1371\/journal.pone.0269153","type":"journal-article","created":{"date-parts":[[2022,6,10]],"date-time":"2022-06-10T17:28:04Z","timestamp":1654882084000},"page":"e0269153","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":12,"title":["Mucosal immunity of mannose-modified chitosan microspheres loaded with the nontyepable Haemophilus influenzae outer membrane protein P6 in BALB\/c mice"],"prefix":"10.1371","volume":"17","author":[{"given":"Yushuai","family":"Ma","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wanru","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanxia","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haixia","family":"Qiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yueli","family":"Chang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shaoping","family":"Kang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yutuo","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2022,6,10]]},"reference":[{"key":"pone.0269153.ref001","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1097\/QCO.0000000000000056","article-title":"Haemophilus influenzae: recent advances in the understanding of molecular pathogenesis and polymicrobial infections","volume":"27","author":"F Jalalvand","year":"2014","journal-title":"Curr Opin Infect Dis"},{"key":"pone.0269153.ref002","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1016\/j.tim.2018.05.001","article-title":"Nontypeable Haemophilus influenzae (NTHi)","volume":"26","author":"LO Bakaletz","year":"2018","journal-title":"Trends Microbiol"},{"key":"pone.0269153.ref003","first-page":"413","article-title":"Haemophilus influenzae type b (Hib) vaccination position paper\u2013July 2013","volume":"88","author":"WHO","year":"2013","journal-title":"Wkly Epidemiol Rec"},{"key":"pone.0269153.ref004","doi-asserted-by":"crossref","first-page":"396","DOI":"10.3201\/eid2303.161552","article-title":"Epidemiology of invasive Haemophilus influenzae disease, Europe, 2007\u20132014","volume":"23","author":"R Whittaker","year":"2017","journal-title":"Emerg Infect Dis"},{"key":"pone.0269153.ref005","doi-asserted-by":"crossref","first-page":"1645","DOI":"10.3201\/eid1709.101991","article-title":"Increasing incidence of invasive Haemophilus influenzae disease in adults, Utah, USA","volume":"17","author":"MP Rubach","year":"2011","journal-title":"Emerg Infect Dis"},{"key":"pone.0269153.ref006","doi-asserted-by":"crossref","first-page":"227","DOI":"10.11150\/kansenshogakuzasshi.85.227","article-title":"Nontypeable Haemophilus influenzae (NTHi) epidemiology","volume":"85","author":"K Sunakawa","year":"2011","journal-title":"Kansenshogaku Zasshi"},{"key":"pone.0269153.ref007","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1128\/CVI.00089-15","article-title":"Vaccines for nontypeable Haemophilus influenzae: the future is now","volume":"22","author":"TF Murphy","year":"2015","journal-title":"Clin Vaccine Immunol"},{"key":"pone.0269153.ref008","doi-asserted-by":"crossref","first-page":"243","DOI":"10.2174\/1874285801812010243","article-title":"Nontypeable Haemophilus influenzae septicemia and urinary tract infection associated with renal stone disease","volume":"12","author":"M St\u00e6rk","year":"2018","journal-title":"Open Microbiol J"},{"key":"pone.0269153.ref009","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1128\/iai.54.3.774-779.1986","article-title":"Antigenic characterization of the P6 protein of nontypeable Haemophilus influenzae","volume":"54","author":"TF Murphy","year":"1986","journal-title":"Infect Immun"},{"key":"pone.0269153.ref010","doi-asserted-by":"crossref","first-page":"2658","DOI":"10.1128\/iai.59.8.2658-2663.1991","article-title":"Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms","volume":"59","author":"MB Nelson","year":"1991","journal-title":"Infect Immun"},{"key":"pone.0269153.ref011","doi-asserted-by":"crossref","first-page":"5187","DOI":"10.1128\/iai.64.12.5187-5192.1996","article-title":"Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media","volume":"64","author":"TF DeMaria","year":"1996","journal-title":"Infect Immun"},{"key":"pone.0269153.ref012","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1111\/j.1574-695X.2012.00967.x","article-title":"Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children","volume":"65","author":"MN Khan","year":"2012","journal-title":"FEMS Immunol Med Microbiol"},{"key":"pone.0269153.ref013","doi-asserted-by":"crossref","first-page":"3252","DOI":"10.1128\/JB.00185-13","article-title":"Dual orientation of the outer membrane lipoprotein P6 of nontypeable Haemophilus influenzae","volume":"195","author":"LV Michel","year":"2013","journal-title":"J Bacteriol"},{"key":"pone.0269153.ref014","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0165-5876(02)00076-9","article-title":"Intranasal immunization with recombinant outer membrane protein P6 induces specific immune responses against nontypeable Haemophilus influenzae","volume":"65","author":"M Hotomi","year":"2002","journal-title":"Int J Pediatr Otorhinolaryngol"},{"key":"pone.0269153.ref015","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1038\/nri1777","article-title":"Mucosal vaccines: the promise and the challenge","volume":"6","author":"MR Neutra","year":"2006","journal-title":"Nat Rev Immunol"},{"key":"pone.0269153.ref016","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1111\/j.1751-7915.2011.00316.x","article-title":"Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge","volume":"5","author":"UB Mayr","year":"2012","journal-title":"Microb Biotechnol"},{"key":"pone.0269153.ref017","doi-asserted-by":"crossref","first-page":"1690","DOI":"10.1128\/CVI.00215-13","article-title":"Enhancement of serum and mucosal immune responses to a Haemophilus influenzae type B vaccine by intranasal delivery","volume":"20","author":"S Fernandez","year":"2013","journal-title":"Clin Vaccine Immunol"},{"key":"pone.0269153.ref018","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1016\/j.jconrel.2007.05.018","article-title":"Controlled release of PEI\/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine","volume":"121","author":"X Zhou","year":"2007","journal-title":"J Control Release"},{"key":"pone.0269153.ref019","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1016\/j.biomaterials.2007.12.025","article-title":"The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization","volume":"29","author":"HL Jiang","year":"2008","journal-title":"Biomaterials"},{"key":"pone.0269153.ref020","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1007\/s00253-014-6147-z","article-title":"Mannose-modified chitosan microspheres enhance OprF-OprI-mediated protection of mice against Pseudomonas aeruginosa infection via induction of mucosal immunity","volume":"99","author":"Z Cui","year":"2014","journal-title":"Appl Microbiol Biotechnol"},{"key":"pone.0269153.ref021","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/S0169-409X(01)00171-5","article-title":"Chitosan as a novel nasal delivery system for vaccines","volume":"51","author":"L Illum","year":"2001","journal-title":"Adv Drug Deliv Rev"},{"key":"pone.0269153.ref022","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1016\/j.vaccine.2006.11.034","article-title":"Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination","volume":"25","author":"DA Zaharoff","year":"2007","journal-title":"Vaccine"},{"key":"pone.0269153.ref023","doi-asserted-by":"crossref","first-page":"4552","DOI":"10.4049\/jimmunol.171.9.4552","article-title":"Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program","volume":"171","author":"M Chieppa","year":"2003","journal-title":"J Immunol"},{"key":"pone.0269153.ref024","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1016\/j.ejpb.2014.04.007","article-title":"Mannose derivative and lipid A dually decorated cationic liposomes as an effective cold chain free oral mucosal vaccine adjuvant-delivery system","volume":"88","author":"N Wang","year":"2014","journal-title":"Eur J Pharm Biopharm"},{"key":"pone.0269153.ref025","doi-asserted-by":"crossref","first-page":"2417","DOI":"10.1002\/eji.1830270941","article-title":"The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells","volume":"27","author":"AJ Engering","year":"1997","journal-title":"Eur J Immunol"},{"key":"pone.0269153.ref026","doi-asserted-by":"crossref","first-page":"2426","DOI":"10.1002\/eji.1830270942","article-title":"Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells","volume":"27","author":"MC Tan","year":"1997","journal-title":"Eur J Immunol"},{"key":"pone.0269153.ref027","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.1517\/14712598.4.12.1953","article-title":"Mannose receptor-targeted vaccines","volume":"4","author":"T Keler","year":"2004","journal-title":"Expert Opin Biol Ther"},{"key":"pone.0269153.ref028","volume-title":"Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology","author":"FM Ausubel","year":"1989"},{"key":"pone.0269153.ref029","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1021\/ma00133a003","article-title":"Some chemical and analytical aspects of polysaccharide modifications. III. Formation of branched-chain, soluble chitosan derivatives","volume":"17","author":"M Yalpani","year":"1984","journal-title":"Macromolecules"},{"key":"pone.0269153.ref030","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1006\/abio.1993.1263","article-title":"Quantitative determination of chitosan and the percentage of free amino groups","volume":"211","author":"E Curotto","year":"1993","journal-title":"Anal Biochem"},{"key":"pone.0269153.ref031","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/j.ejpb.2004.05.006","article-title":"In vitro study of the immune stimulating activity of an athrophic rhinitis vaccine associated to chitosan microspheres","volume":"58","author":"HL Jiang","year":"2004","journal-title":"Eur J Pharm Biopharm"},{"key":"pone.0269153.ref032","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1186\/s13054-019-2574-7","article-title":"Interleukin-26 is overexpressed in human sepsis and contributes to inflammation, organ injury, and mortality in murine sepsis","volume":"23","author":"Hongmei Tu","year":"2019","journal-title":"Crit Care"},{"key":"pone.0269153.ref033","doi-asserted-by":"crossref","first-page":"2294","DOI":"10.1128\/IAI.68.4.2294-2300.2000","article-title":"Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization","volume":"68","author":"S Kodama","year":"2000","journal-title":"Infect Immun"},{"key":"pone.0269153.ref034","doi-asserted-by":"crossref","first-page":"2964","DOI":"10.1128\/IAI.69.5.2964-2971.2001","article-title":"Intranasal immunization enhances clearance of nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media","volume":"69","author":"A Sabirov","year":"2001","journal-title":"Infect Immun"},{"key":"pone.0269153.ref035","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1023\/A:1015987516796","article-title":"A novel approach to the oral delivery of micro- or nanoparticles","volume":"6","author":"R Bodmeier","year":"1989","journal-title":"Pharm Res"},{"key":"pone.0269153.ref036","first-page":"119","article-title":"The use of shells made of poly(ethylene glycol) and chitosan to ensure the biocompatibility of nanoparticles in biomedical applications","volume":"44","author":"T. Kubiak","year":"2014","journal-title":"Polim Med"},{"key":"pone.0269153.ref037","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.carbpol.2017.07.058","article-title":"Preparation and biological activity studies of resveratrol loaded ionically cross-linked chitosan-TPP nanoparticles","volume":"175","author":"J Wu","year":"2017","journal-title":"Carbohydr Polym"},{"key":"pone.0269153.ref038","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/S0378-5173(02)00486-6","article-title":"Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo","volume":"249","author":"Y Pan","year":"2002","journal-title":"Int J Pharm"},{"key":"pone.0269153.ref039","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.ijpharm.2005.09.013","article-title":"Nasal administration of carbamazepine using chitosan microspheres: in vitro\/in vivo studies","volume":"307","author":"E Gavini","year":"2006","journal-title":"Int J Pharm"},{"key":"pone.0269153.ref040","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.ejpb.2006.01.010","article-title":"In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT","volume":"63","author":"ML Kang","year":"2006","journal-title":"Eur J Pharm Biopharm"},{"key":"pone.0269153.ref041","first-page":"97","article-title":"Chitosan-based intranasal vaccine against Escherichia coli O157:H7","volume":"20","author":"T Doavi","year":"2016","journal-title":"Iran Biomed J"},{"key":"pone.0269153.ref042","doi-asserted-by":"crossref","first-page":"e190","DOI":"10.1016\/j.jconrel.2011.08.082","article-title":"Preparation and evaluation of mannose receptor mediated macrophage targeting delivery system","volume":"152","author":"L Zhu","year":"2011","journal-title":"J Control Release"},{"key":"pone.0269153.ref043","doi-asserted-by":"crossref","first-page":"445","DOI":"10.3389\/fcimb.2017.00445","article-title":"Intranasal vaccination with mannosylated chitosan formulated DNA vaccine enables robust IgA and cellular response induction in the lungs of mice and improves protection against pulmonary mycobacterial challenge","volume":"7","author":"M Wu","year":"2017","journal-title":"Front Cell Infect Microbiol"},{"key":"pone.0269153.ref044","doi-asserted-by":"crossref","first-page":"1860","DOI":"10.3389\/fimmu.2018.01860","article-title":"CD4 T(RM) cells following infection and immunization: Implications for more effective vaccine design","volume":"9","author":"MM Wilk","year":"2018","journal-title":"Front Immunol"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0269153","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,10]],"date-time":"2022-06-10T17:28:45Z","timestamp":1654882125000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0269153"}},"subtitle":[],"editor":[{"given":"Ray","family":"Borrow","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2022,6,10]]},"references-count":44,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6,10]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0269153","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,10]]}}}