Evaluation of the reactogenicity, adjuvanticity and antigenicity of LT(R192G) and LT(R192G/L211A) by intradermal immunization in mice

Autoři: Milton Maciel, Jr. aff001;  Mark Smith aff004;  Steven T. Poole aff001;  Renee M. Laird aff001;  Julianne E. Rollenhagen aff001;  Robert W. Kaminski aff004;  Heather Wenzel aff005;  A. Louis Bourgeois aff005;  Stephen J. Savarino aff003
Působiště autorů: Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, United States of America aff001;  Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America aff002;  Enteric Diseases Department, Naval Medical Research Center, Silver Spring, MD, United States of America aff003;  Subunit Enteric Vaccines and Immunology, Walter Reed Army Institute of Research, Silver Spring, MD, United States of America aff004;  PATH, Washington, D.C., United States of America aff005;  Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America aff006
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224073


The development of an effective subunit vaccine is frequently complicated by the difficulty of eliciting protective immune responses, often requiring the co-administration of an adjuvant. Heat-labile toxin (LT), an enterotoxin expressed by enterotoxigenic E. coli (ETEC) with an AB5 structure similar to cholera toxin, is a strong adjuvant. While the mucosa represents the natural route of exposure to LT and related toxins, the clinical utility of LT and similar adjuvants given by mucosal routes has been limited by toxicity, as well as the association between intranasal delivery of LT and Bell’s palsy. Single and double amino acid mutants of LT, LT(R192G)/mLT and LT(R192G/L211A)/dmLT respectively, have been proposed as alternatives to reduce the toxicity associated with the holotoxin. In the present study, we compared mLT and dmLT given via a non-mucosal route (i.e. intradermally) to investigate their adjuvanticity when co-administrated with an enterotoxigenic E. coli vaccine candidate, CfaEB. Antigenicity (i.e. ability to elicit response against LT) and reactogenicity at the injection site were also evaluated. BALB/c mice were immunized by the intradermal route with CfaEB plus increasing doses of either mLT or dmLT (0.01 to 2.5 μg). Both adjuvants induced dose-dependent skin reactogenicity, with dmLT being less reactogenic than mLT. Both adjuvants significantly boosted the anti-CfaE IgG and functional hemagglutination inhibiting (HAI) antibody responses, compared to the antigen alone. In addition to inducing anti-LT responses, even at the lowest dose tested (0.01 μg), the adjuvants also prompted in vitro cytokine responses (IFN-γ, IL-4, IL-5, IL-10 and IL-17) that followed different patterns, depending on the protein used for stimulation (CfaE or LTB) and/or the dose used for immunization. The two LT mutants evaluated here, mLT and dmLT, are potent adjuvants for intradermal immunization and should be further investigated for the intradermal delivery of subunit ETEC vaccines.

Klíčová slova:

Antibodies – Antigens – Cytokines – Immune response – Immunologic adjuvants – Vaccines – Erythema


1. Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland. N Engl J Med. 2004;350(9):896–903. doi: 10.1056/NEJMoa030595 14985487.

2. da Hora VP, Conceicao FR, Dellagostin OA, Doolan DL. Non-toxic derivatives of LT as potent adjuvants. Vaccine. 2011;29(8):1538–44. doi: 10.1016/j.vaccine.2010.11.091 21163247.

3. Rappuoli R, Pizza M, Douce G, Dougan G. <LT structure & adjuvanticity_IT_1999pdf.pdf>. Immunology Totay. 1999;20(11):493–500.

4. Norton EB, Lawson LB, Freytag LC, Clements JD. Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant. Clinical and vaccine immunology: CVI. 2011;18(4):546–51. doi: 10.1128/CVI.00538-10 21288994; PubMed Central PMCID: PMC3122563.

5. Sincock SA, Hall ER, Woods CM, O'Dowd A, Poole ST, McVeigh AL, et al. Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates. Vaccine. 2016;34(2):284–91. doi: 10.1016/j.vaccine.2015.11.017 26597148.

6. Holmgren J, Bourgeois L, Carlin N, Clements J, Gustafsson B, Lundgren A, et al. Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant. Vaccine. 2013;31(20):2457–64. doi: 10.1016/j.vaccine.2013.03.027 23541621.

7. Lundgren A, Bourgeois L, Carlin N, Clements J, Gustafsson B, Hartford M, et al. Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind, randomized, placebo-controlled Phase I study. Vaccine. 2014;32(52):7077–84. doi: 10.1016/j.vaccine.2014.10.069 25444830.

8. Lapa JA, Sincock SA, Ananthakrishnan M, Porter CK, Cassels FJ, Brinkley C, et al. Randomized clinical trial assessing the safety and immunogenicity of oral microencapsulated enterotoxigenic Escherichia coli surface antigen 6 with or without heat-labile enterotoxin with mutation R192G. Clinical and vaccine immunology: CVI. 2008;15(8):1222–8. doi: 10.1128/CVI.00491-07 18579693; PubMed Central PMCID: PMC2519302.

9. Akhtar M, Chowdhury MI, Bhuiyan TR, Kaim J, Ahmed T, Rafique TA, et al. Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses. Vaccine. 2018. doi: 10.1016/j.vaccine.2018.11.040 30473185.

10. Dickinson BL, Clemens JD. Dissociation of Escherichia coli Heat-Labile Enterotoxin Adjuvanticity from ADP-Ribosyltransferase Activity. Infection and immunity. 1995;63(5):1617–23. 7729864

11. Behrens RH, Cramer JP, Jelinek T, Shaw H, von Sonnenburg F, Wilbraham D, et al. Efficacy and safety of a patch vaccine containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: a phase 3, randomised, double-blind, placebo-controlled field trial in travellers from Europe to Mexico and Guatemala. The Lancet Infectious Diseases. 2014;14(3):197–204. doi: 10.1016/S1473-3099(13)70297-4 24291168

12. Poole ST, McVeigh AL, Anantha RP, Lee LH, Akay YM, Pontzer EA, et al. Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway. Mol Microbiol. 2007;63(5):1372–84. doi: 10.1111/j.1365-2958.2007.05612.x 17302815.

13. Poole ST, Maciel M Jr., Dinadayala P, Dori KE, McVeigh AL, Liu Y, et al. Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates. Infection and immunity. 2019;87(3). doi: 10.1128/IAI.00788-18 30602504.

14. Rollenhagen JE, Jones F, Hall E, Maves R, Nunez G, Espinosa N, et al. Establishment, validation and application of a New World Primate model of ETEC disease for vaccine development. Infection and immunity. 2018. doi: 10.1128/IAI.00634-18 30510102; PubMed Central PMCID: PMC6346130.

15. Savarino SJ, McKenzie R, Tribble D, Porter C, O'Dowd A, Cantrell JA, et al. Prophylactic Efficacy of Hyperimmune Bovine Colostral Antiadhesin Antibodies Against Enterotoxigenic Escherichia coli Diarrhea: A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Trial. Journal of Infectious Diseases. 2017. doi: 10.1093/infdis/jix144 28541500

16. Harro C, Gutierrez RL, Talaat K, Porter C, Riddle MS, Maciel M, Jr., et al., editors. Protective efficacy of an enterotoxigenic E. coli fimbrial tip adhesin vaccine given with LTR192G by intradermal vaccination against experimental challenge with CFA/I-ETEC in adult volunteers. 50th US-Japan Cooperative Medical Sciences Program Joint Panel Conference on Cholera and Other Bacterial Enteric Infections; 2016; Bethesda, MD. Bethesda, MD2016.

17. Kim YC, Jarrahian C, Zehrung D, Mitragotri S, Prausnitz MR. Delivery systems for intradermal vaccination. Curr Top Microbiol Immunol. 2012;351:77–112. doi: 10.1007/82_2011_123 21472533; PubMed Central PMCID: PMC3173582.

18. Nelson KS, Janssen JM, Troy SB, Maldonado Y. Intradermal fractional dose inactivated polio vaccine: a review of the literature. Vaccine. 2012;30(2):121–5. doi: 10.1016/j.vaccine.2011.11.018 22100886.

19. Hung IFN, Yuen KY. Immunogenicity, safety and tolerability of intradermal influenza vaccines. Human vaccines & immunotherapeutics. 2018;14(3):565–70. doi: 10.1080/21645515.2017.1328332 28604266; PubMed Central PMCID: PMC5861844.

20. Combadiere B, Liard C. Transcutaneous and intradermal vaccination. Human vaccines. 2011;7(8):811–27. doi: 10.4161/hv.7.8.16274 21817854.

21. Lambert PH, Laurent PE. Intradermal vaccine delivery: will new delivery systems transform vaccine administration? Vaccine. 2008;26(26):3197–208. doi: 10.1016/j.vaccine.2008.03.095 18486285.

22. Belyakov IM, Hammond SA, Ahlers JD, Glenn GM, Berzofsky JA. Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells. Journal of Clinical Investigation. 2004;113(7):998–1007. doi: 10.1172/JCI20261 15057306

23. Chang SY, Cha HR, Igarashi O, Rennert PD, Kissenpfennig A, Malissen B, et al. Cutting Edge: Langerin+ Dendritic Cells in the Mesenteric Lymph Node Set the Stage for Skin and Gut Immune System Cross-Talk. The Journal of Immunology. 2008;180(7):4361–5. doi: 10.4049/jimmunol.180.7.4361 18354155

24. Zoeteweij JP, Epperson DE, Porter JD, Zhang CX, Frolova OY, Constantinides AP, et al. GM1 Binding-Deficient Exotoxin Is a Potent Noninflammatory Broad Spectrum Intradermal Immunoadjuvant. The Journal of Immunology. 2006;177(2):1197–207. doi: 10.4049/jimmunol.177.2.1197 16818778

25. Guidry JJ, Cardenas L, Cheng E, Clements JD. Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin. Infection and immunity. 1997;65(12):4943–50. Epub 4943. 9393780; PubMed Central PMCID: PMC175713.

26. White JA, Haghighi C, Brunner J, Estrada M, Lal M, Chen D. Preformulation studies with the Escherichia coli double mutant heat-labile toxin adjuvant for use in an oral vaccine. Journal of immunological methods. 2017;451:83–9. doi: 10.1016/j.jim.2017.09.003 28939395; PubMed Central PMCID: PMC5703769.

27. Li YF, Poole S, Nishio K, Jang K, Rasulova F, McVeigh A, et al. Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(26):10793–8. doi: 10.1073/pnas.0812843106 19515814; PubMed Central PMCID: PMC2705562.

28. Draize J, Woodard G, Calvery H. Mwethods for the study of irritation and toxicity of substances applied topically to the skin and muous membranes. Journal of Pharmacology and Experimental Therpeutics. 1944;82:377–90.

29. Li YF, Poole S, Rasulova F, Esser L, Savarino SJ, Xia D. Crystallization and preliminary X-ray diffraction analysis of CfaE, the adhesive subunit of the CFA/I fimbriae from human enterotoxigenic Escherichia coli. Acta crystallographica Section F, Structural biology and crystallization communications. 2006;62(Pt 2):121–4. doi: 10.1107/S1744309105043198 16511280; PubMed Central PMCID: PMC2150950.

30. Anantha RP, McVeigh AL, Lee LH, Agnew MK, Cassels FJ, Scott DA, et al. Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli. Infection and immunity. 2004;72(12):7190–201. doi: 10.1128/IAI.72.12.7190-7201.2004 15557644; PubMed Central PMCID: PMC529125.

31. Chen X, Wu MX. Laser vaccine adjuvant for cutaneous immunization. Expert review of vaccines. 2011;10(10):1397–403. doi: 10.1586/erv.11.112 21988305; PubMed Central PMCID: PMC3250349.

32. Oreskovic Z, Nechvatalova K, Krejci J, Kummer V, Faldyna M. Aspects of intradermal immunization with different adjuvants: The role of dendritic cells and Th1/Th2 response. PloS one. 2019;14(2):e0211896. doi: 10.1371/journal.pone.0211896 30742635; PubMed Central PMCID: PMC6370205.

33. Carter D, van Hoeven N, Baldwin S, Levin Y, Kochba E, Magill A, et al. The adjuvant GLA-AF enhances human intradermal vaccine responses. Sci Adv. 2018;4(9):eaas9930. doi: 10.1126/sciadv.aas9930 30221194; PubMed Central PMCID: PMC6136895.

34. Clements JD, Norton EB. The Mucosal Vaccine Adjuvant LT(R192G/L211A) or dmLT. mSphere. 2018;3(4). doi: 10.1128/mSphere.00215-18 30045966; PubMed Central PMCID: PMC6060342.

35. Heine SJ, Diaz-McNair J, Andar AU, Drachenberg CB, van de Verg L, Walker R, et al. Intradermal delivery of Shigella IpaB and IpaD type III secretion proteins: kinetics of cell recruitment and antigen uptake, mucosal and systemic immunity, and protection across serotypes. Journal of immunology. 2014;192(4):1630–40. doi: 10.4049/jimmunol.1302743 24453241; PubMed Central PMCID: PMC3998105.

36. Maciel M Jr., Bauer D, Baudier RL, Bitoun J, Clements JD, Poole ST, et al. Intradermal or sublingual delivery and heat-labile enterotoxin (LT) proteins shape immunologic outcomes to CFA/I fimbriae-derived subunit antigen vaccine against enterotoxigenic E. coli. Infection and immunity. 2019. doi: 10.1128/IAI.00460-19 31427449.

37. Luo Q, Vickers TJ, Fleckenstein JM. Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin. Clinical and vaccine immunology: CVI. 2016;23(7):628–37. doi: 10.1128/CVI.00248-16 27226279; PubMed Central PMCID: PMC4933781.

38. Liang H, Poncet D, Seydoux E, Rintala ND, Maciel M Jr., Ruiz S, et al. The TLR4 agonist adjuvant SLA-SE promotes functional mucosal antibodies against a parenterally delivered ETEC vaccine. NPJ Vaccines. 2019;4:19. doi: 10.1038/s41541-019-0116-6 31149350; PubMed Central PMCID: PMC6538625.

39. Norton EB, Maciel M Jr., Baudier R, Cunningham CK, Janikowski U, Laird RM, et al., editors. Detailed LT antibody analysis on ETEC clinical trial samples. 8th Vaccines for Enteric Diseases; 2015; Edinburgh, UK.

40. Maciel M Jr., Baudier R, Valli E, Mulloy CD, Porter C, Gutierrez RL, et al., editors. Optimizing LT Antibody Analyses to Predict Protection from Oral H10407 ETEC Challenge. 9th Vaccines Against Enteric Diseases; 2017 9-11th October; Albufeira, Portugal.

41. Norton EB, Lawson LB, Mahdi Z, Freytag LC, Clements JD. The A subunit of Escherichia coli heat-labile enterotoxin functions as a mucosal adjuvant and promotes IgG2a, IgA, and Th17 responses to vaccine antigens. Infection and immunity. 2012;80(7):2426–35. doi: 10.1128/IAI.00181-12 22526674; PubMed Central PMCID: PMC3416479.

42. Leach S, Clements JD, Kaim J, Lundgren A. The adjuvant double mutant Escherichia coli heat labile toxin enhances IL-17A production in human T cells specific for bacterial vaccine antigens. PloS one. 2012;7(12):e51718. doi: 10.1371/journal.pone.0051718 23284753; PubMed Central PMCID: PMC3527457.

43. Mitsdoerffer M, Lee Y, Jager A, Kim HJ, Korn T, Kolls JK, et al. Proinflammatory T helper type 17 cells are effective B-cell helpers. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(32):14292–7. doi: 10.1073/pnas.1009234107 20660725; PubMed Central PMCID: PMC2922571.

44. Kuwabara T, Ishikawa F, Kondo M, Kakiuchi T. The Role of IL-17 and Related Cytokines in Inflammatory Autoimmune Diseases. Mediators Inflamm. 2017;2017:3908061. doi: 10.1155/2017/3908061 28316374; PubMed Central PMCID: PMC5337858.

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2019 Číslo 11