Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®-mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Department of Analytical Chemistry University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Microbiology and Parasitology Institute of Tropical Health University of Navarra 31008 Pamplona Spain

Department of Nuclear Medicine Clínica Universidad de Navarra 31008 Pamplona Spain

Department of Pharmacy and Pharmaceutical Technology University of Navarra 31008 Pamplona Spain

Zobrazit více v PubMed

Kaper J.B., Nataro J.P., Mobley H.L.T. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2004;2:123–140. doi: 10.1038/nrmicro818. PubMed DOI

Fleckenstein J.M., Hardwidge P.R., Munson G.P., Rasko D.A., Sommerfelt H., Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect. 2010;12:89–98. doi: 10.1016/j.micinf.2009.10.002. PubMed DOI PMC

Khalil I.A., Troeger C., Blacker B.F., Rao P.C., Brown A., Atherly D.E., Brewer T.G., Engmann C.M., Houpt E.R., Kang G., et al. Morbidity and mortality due to shigella and Enterotoxigenic Escherichia coli diarrhoea: The Global Burden of Disease Study 1990–2016. Lancet Infect. Dis. 2018;18:1229–1240. doi: 10.1016/S1473-3099(18)30475-4. PubMed DOI PMC

Sanders J.W., Riddle M.S., Taylor D.N., DuPont H.L. Travel Medicine. Elsevier Ltd.; Amsterdam, The Netherlands: 2008. Epidemiology of Travelers’ Diarrhea; pp. 187–198.

Svennerholm A.-M. From cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development. Indian J. Med. Res. 2011;133:188–196. PubMed PMC

Fleckenstein J.M., Kuhlmann F.M. Enterotoxigenic Escherichia coli Infections. Curr. Infect. Dis. Rep. 2019;21:9. doi: 10.1007/s11908-019-0665-x. PubMed DOI PMC

Fleckenstein J.M. Confronting Challenges to Enterotoxigenic Escherichia coli Vaccine Development. Front. Trop. Dis. 2021;2:9907. doi: 10.3389/fitd.2021.709907. PubMed DOI PMC

Li M., Zhou H., Yang C., Wu Y., Zhou X., Liu H., Wang Y. Bacterial outer membrane vesicles as a platform for biomedical applications: An update. J. Control. Release. 2020;323:253–268. doi: 10.1016/j.jconrel.2020.04.031. PubMed DOI

Pollard A.J., Bijker E.M. A guide to vaccinology: From basic principles to new developments. Nat. Rev. Immunol. 2021;21:83–100. doi: 10.1038/s41577-020-00479-7. PubMed DOI PMC

Bai X., Findlow J., Borrow R. Recombinant protein meningococcal serogroup B vaccine combined with outer membrane vesicles. Expert Opin. Biol. Ther. 2011;11:969–985. doi: 10.1517/14712598.2011.585965. PubMed DOI

Gorringe A.R., Pajón R. Bexsero: A multicomponent vaccine for prevention of meningococcal disease. Hum. Vaccin. Immunother. 2012;8:174–183. doi: 10.4161/hv.18500. PubMed DOI

Li Y., Jin L., Chen T. The Effects of Secretory IgA in the Mucosal Immune System. Biomed Res. Int. 2020;2020:2032057. doi: 10.1155/2020/2032057. PubMed DOI PMC

Vela Ramirez J.E., Sharpe L.A., Peppas N.A. Current state and challenges in developing oral vaccines. Adv. Drug Deliv. Rev. 2017;114:116–131. doi: 10.1016/j.addr.2017.04.008. PubMed DOI PMC

Forssell H. Gastric mucosal defence mechanisms: A brief review. Scand. J. Gastroenterol. Suppl. 1988;155:23–28. doi: 10.3109/00365528809096277. PubMed DOI

Jazayeri S.D., Lim H.X., Shameli K., Yeap S.K., Poh C.L. Nano and Microparticles as Potential Oral Vaccine Carriers and Adjuvants Against Infectious Diseases. Front. Pharmacol. 2021;12:2286. doi: 10.3389/fphar.2021.682286. PubMed DOI PMC

Duan Q., Xia P., Nandre R., Zhang W., Zhu G. Review of Newly Identified Functions Associated with the Heat-Labile Toxin of Enterotoxigenic Escherichia coli. Front. Cell. Infect. Microbiol. 2019;9:292. doi: 10.3389/fcimb.2019.00292. PubMed DOI PMC

Zhang C., Iqbal J., Gómez-Duarte O.G. Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization. Vet. Microbiol. 2017;202:90–100. doi: 10.1016/j.vetmic.2016.02.001. PubMed DOI PMC

Luo Q., Qadri F., Kansal R., Rasko D.A., Sheikh A., Fleckenstein J.M. Conservation and Immunogenicity of Novel Antigens in Diverse Isolates of Enterotoxigenic Escherichia coli. PLoS Negl. Trop. Dis. 2015;9:e0003446. doi: 10.1371/journal.pntd.0003446. PubMed DOI PMC

Rao M.R., Wierzba T.F., Savarino S.J., Abu-Elyazeed R., El-Ghoreb N., Hall E.R., Naficy A., Abdel-Messih I., Frenck R.W., Jr., Svennerholm A., et al. Serologic Correlates of Protection against Enterotoxigenic Escherichia coli Diarrhea. J. Infect. Dis. 2005;191:562–570. doi: 10.1086/427662. PubMed DOI

Gaastra W., Svennrholm A.-M. Colonization factors of human enterotoxigenic Escherichia coli (ETEC) Trends Microbiol. 1996;4:444–452. doi: 10.1016/0966-842X(96)10068-8. PubMed DOI

Irache J.M., González-Navarro C.J. Zein nanoparticles as vehicles for oral delivery purposes. Nanomedicine. 2017;12:1209–1211. doi: 10.2217/nnm-2017-0075. PubMed DOI

Berzosa M., Pastor Y., Irache J.M., Gamazo C. Applications of Nanobiotechnology for Neglected Tropical Diseases. Elsevier Ltd.; Amsterdam, The Netherlands: 2021. Experimental vaccination with nanoparticles containing Escherichia coli virulence factors; pp. 3–27.

Inchaurraga L., Martínez-López A.L., Abdulkarim M., Gumbleton M., Quincoces G., Peñuelas I., Martin-Arbella N., Irache J.M. Modulation of the fate of zein nanoparticles by their coating with a Gantrez® AN-thiamine polymer conjugate. Int. J. Pharm. X. 2019;1:100006. doi: 10.1016/j.ijpx.2019.100006. PubMed DOI PMC

Tamayo I., Irache J.M., Mansilla C., Ochoa-Repáraz J., Lasarte J.J., Gamazo C. Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation. Clin. Vaccine Immunol. 2010;17:1356–1362. doi: 10.1128/CVI.00164-10. PubMed DOI PMC

Zhang C., Shi G., Zhang J., Song H., Niu J., Shi S., Huang P., Wang Y., Wang W., Li C., et al. Targeted antigen delivery to dendritic cell via functionalized alginate nanoparticles for cancer immunotherapy. J. Control. Release. 2017;256:170–181. doi: 10.1016/j.jconrel.2017.04.020. PubMed DOI

Lachén-Montes M., González-Morales A., Zelaya M.V., Pérez-Valderrama E., Ausín K., Ferrer I., Fernández-Irigoyen J., Santamaría E. Olfactory bulb neuroproteomics reveals a chronological perturbation of survival routes and a disruption of prohibitin complex during Alzheimer’s disease progression. Sci. Rep. 2017;7:9115. doi: 10.1038/s41598-017-09481-x. PubMed DOI PMC

Lachén-Montes M., González-Morales A., de Morentin X.M., Pérez-Valderrama E., Ausín K., Zelaya M.V., Serna A., Aso E., Ferrer I., Fernández-Irigoyen J., et al. An early dysregulation of FAK and MEK/ERK signaling pathways precedes the β-amyloid deposition in the olfactory bulb of APP/PS1 mouse model of Alzheimer’s disease. J. Proteom. 2016;148:149–158. doi: 10.1016/j.jprot.2016.07.032. PubMed DOI

Shilov I.V., Seymour S.L., Patel A.A., Loboda A., Tang W.H., Keating S.P., Hunter C.L., Nuwaysir L.M., Schaeffer D.A. The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra. Mol. Cell. Proteom. 2007;6:1638–1655. doi: 10.1074/mcp.T600050-MCP200. PubMed DOI

Tang W.H., Shilov I.V., Seymour S.L. Nonlinear Fitting Method for Determining Local False Discovery Rates from Decoy Database Searches. J. Proteome Res. 2008;7:3661–3667. doi: 10.1021/pr070492f. PubMed DOI

Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 1979;76:4350–4354. doi: 10.1073/pnas.76.9.4350. PubMed DOI PMC

Brotons-Canto A., Gamazo C., Martín-Arbella N., Abdulkarim M., Matías J., Gumbleton M., Irache J.M. Evaluation of nanoparticles as oral vehicles for immunotherapy against experimental peanut allergy. Int. J. Biol. Macromol. 2018;110:328–335. doi: 10.1016/j.ijbiomac.2017.09.109. PubMed DOI

Matías J., Brotons A., Cenoz S., Pérez I., Abdulkarim M., Gumbleton M., Irache J.M., Gamazo C. Oral Immunogenicity in Mice and Sows of Enterotoxigenic Escherichia coli Outer-Membrane Vesicles Incorporated into Zein-Based Nanoparticles. Vaccines. 2019;8:11. doi: 10.3390/vaccines8010011. PubMed DOI PMC

Zhang W., Sack D.A. Progress and hurdles in the development of vaccines against Enterotoxigenic Escherichia coli in humans. Expert Rev. Vaccines. 2012;11:677–694. doi: 10.1586/erv.12.37. PubMed DOI

Leach S., Lundgren A., Carlin N., Löfstrand M., Svennerholm A.-M. Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent Enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. Vaccine. 2017;35:3966–3973. doi: 10.1016/j.vaccine.2017.06.006. PubMed DOI

Seo H., Garcia C., Ruan X., Duan Q., Sack D.A., Zhang W. Preclinical Characterization of Immunogenicity and Efficacy against Diarrhea from MecVax, a Multivalent Enterotoxigenic E. coli Vaccine Candidate. Infect. Immun. 2021;89 doi: 10.1128/IAI.00106-21. PubMed DOI PMC

Qadri F., Akhtar M., Bhuiyan T.R., Chowdhury M.I., Ahmed T., Rafique T.A., Khan A., Rahman S.I.A., Khanam F., Lundgren A., et al. Safety and immunogenicity of the oral, inactivated, Enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi children and infants: A double-blind, randomised, placebo-controlled phase 1/2 trial. Lancet Infect. Dis. 2020;20:208–219. doi: 10.1016/S1473-3099(19)30571-7. PubMed DOI PMC

Khalil I., Walker R., Porter C.K., Muhib F., Chilengi R., Cravioto A., Guerrant R., Svennerholm A.M., Qadri F., Baqar S., et al. Enterotoxigenic Escherichia coli (ETEC) vaccines: Priority activities to enable product development, licensure, and global access. Vaccine. 2021;39:4266–4277. doi: 10.1016/j.vaccine.2021.04.018. PubMed DOI PMC

van der Pol L., Stork M., van der Ley P. Outer membrane vesicles as platform vaccine technology. Biotechnol. J. 2015;10:1689–1706. doi: 10.1002/biot.201400395. PubMed DOI PMC

Chen L., Valentine J.L., Huang C.-J., Endicott C.E., Moeller T.D., Rasmussen J.A., Fletcher J.R., Boll J.M., Rosenthal J.A., Dobruchowska J., et al. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies. Proc. Natl. Acad. Sci. USA. 2016;113:E3609–E3618. doi: 10.1073/pnas.1518311113. PubMed DOI PMC

Isidean S.D., Riddle M.S., Savarino S.J., Porter C.K. A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression. Vaccine. 2011;29:6167–6178. doi: 10.1016/j.vaccine.2011.06.084. PubMed DOI

Vidal R.M., Muhsen K., Tennant S.M., Svennerholm A.M., Sow S.O., Sur D., Zaidi A.K.M., Faruque A.S.G., Saha D., Adegbola R., et al. Colonization factors among Enterotoxigenic Escherichia coli isolates from children with moderate-to-severe diarrhea and from matched controls in the Global Enteric Multicenter Study (GEMS) PLoS Negl. Trop. Dis. 2019;13:e0007037. doi: 10.1371/journal.pntd.0007037. PubMed DOI PMC

Kuhlmann F.M., Martin J., Hazen T.H., Vickers T.J., Pashos M., Okhuysen P.C., Gómez-Duarte O.G., Cebelinski E., Boxrud D., Del Canto F., et al. Conservation and global distribution of noncanonical antigens in Enterotoxigenic Escherichia coli. PLoS Negl. Trop. Dis. 2019;13:e0007825. doi: 10.1371/journal.pntd.0007825. PubMed DOI PMC

Qadri F., Das S.K., Faruque A.S.G., Fuchs G.J., Albert M.J., Sack R.B., Svennerholm A.M. Prevalence of toxin types and colonization factors in Enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Bangladesh. J. Clin. Microbiol. 2000;38:27–31. doi: 10.1128/JCM.38.1.27-31.2000. PubMed DOI PMC

Zhang S., Wu Q., Zhang J., Lai Z., Zhu X. Prevalence, genetic diversity, and antibiotic resistance of Enterotoxigenic Escherichia coli in retail ready-to-eat foods in China. Food Control. 2016;68:236–243. doi: 10.1016/j.foodcont.2016.03.051. DOI

Guerra J.A., Romero-Herazo Y.C., Arzuza O., Gómez-Duarte O.G. Phenotypic and genotypic characterization of Enterotoxigenic Escherichia coli clinical isolates from Northern Colombia, South America. BioMed Res. Int. 2014;2014:236260. doi: 10.1155/2014/236260. PubMed DOI PMC

Harris J.A., Roy K., Woo-Rasberry V., Hamilton D.J., Kansal R., Qadri F., Fleckenstein J.M. Directed evaluation of Enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates. PLoS Negl. Trop. Dis. 2011;5:e1428. doi: 10.1371/journal.pntd.0001428. PubMed DOI PMC

Rivas Z.P., Talbot K.M., Merselis L.C., McCormack R.M., Adkins B., Munson G.P. CexE Is a Coat Protein and Virulence Factor of Diarrheagenic Pathogens. Front. Microbiol. 2020;11:626–638. doi: 10.3389/fmicb.2020.01374. PubMed DOI PMC

Chakraborty S., Randall A., Vickers T.J., Molina D., Harro C.D., DeNearing B., Brubaker J., Sack D.A., Bourgeois A.L., Felgner P.L., et al. Human Experimental Challenge with Enterotoxigenic Escherichia coli Elicits Immune Responses to Canonical and Novel Antigens Relevant to Vaccine Development. J. Infect. Dis. 2018;218:1436–1446. doi: 10.1093/infdis/jiy312. PubMed DOI PMC

De Smet R., Allais L., Cuvelier C.A. Recent advances in oral vaccine development. Hum. Vaccin. Immunother. 2014;10:1309–1318. doi: 10.4161/hv.28166. PubMed DOI PMC

Taddio A., Ipp M., Thivakaran S., Jamal A., Parikh C., Smart S., Sovran J., Stephens D., Katz J. Survey of the prevalence of immunization non-compliance due to needle fears in children and adults. Vaccine. 2012;30:4807–4812. doi: 10.1016/j.vaccine.2012.05.011. PubMed DOI

Davitt C.J.H., Lavelle E.C. Delivery strategies to enhance oral vaccination against enteric infections. Adv. Drug Deliv. Rev. 2015;91:52–69. doi: 10.1016/j.addr.2015.03.007. PubMed DOI

Levine M.M., Dougan G. Optimism over vaccines administered via mucosal surfaces. Lancet. 1998;351:1375–1376. doi: 10.1016/S0140-6736(05)79439-3. PubMed DOI

Mudie D.M., Amidon G.L., Amidon G.E. Physiological Parameters for Oral Delivery and in Vitro Testing. Mol. Pharm. 2010;7:1388–1405. doi: 10.1021/mp100149j. PubMed DOI PMC

Wilson-Welder J.H., Torres M.P., Kipper M.J., Mallapragada S.K., Wannemuehler M.J., Narasimhan B. Vaccine adjuvants: Current challenges and future approaches. J. Pharm. Sci. 2009;98:1278–1316. doi: 10.1002/jps.21523. PubMed DOI PMC

Kunisawa J., Kurashima Y., Kiyono H. Gut-associated lymphoid tissues for the development of oral vaccines. Adv. Drug Deliv. Rev. 2012;64:523–530. doi: 10.1016/j.addr.2011.07.003. PubMed DOI

McNeela E.A., Lavelle E.C. Recent Advances in Microparticle and Nanoparticle Delivery Vehicles for Mucosal Vaccination. Curr. Top. Microbiol. Immunol. 2012;354:75–99. doi: 10.1007/82_2011_140. PubMed DOI

Mann J.F., Acevedo R., del Campo J., Pérez O., Ferro V.A. Delivery systems: A vaccine strategy for overcoming mucosal tolerance? Expert Rev. Vaccines. 2009;8:103–112. doi: 10.1586/14760584.8.1.103. PubMed DOI

Camacho A.I., Da Costa Martins R., Tamayo I., de Souza J., Lasarte J.J., Mansilla C., Esparza I., Irache J.M., Gamazo C. Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators. Vaccine. 2011 doi: 10.1016/j.vaccine.2011.05.072. PubMed DOI

Salman H.H., Gamazo C., Campanero M.A., Irache J.M. Bioadhesive Mannosylated Nanoparticles for Oral Drug Delivery. J. Nanosci. Nanotechnol. 2006;6:3203–3209. doi: 10.1166/jnn.2006.445. PubMed DOI

Matías J., Pastor Y., Irache J.M., Gamazo C. Protective Passive Immunity in Escherichia coli ETEC-Challenged Neonatal Mice Conferred by Orally Immunized Dams with Nanoparticles Containing Homologous Outer Membrane Vesicles. Vaccines. 2020;8:286. doi: 10.3390/vaccines8020286. PubMed DOI PMC