Intramuscular immunization of mice with the recombinant enterotoxigenic Escherichia coli (ETEC) antigen CfaEB, adjuvanted with the synthetic Toll-like receptor 4 (TLR4) agonist SLA in a stable emulsion (SLA-SE), was shown in 2019 to elicit functional mucosal antibodies in the intestine, with synergy between SLA-SE and the detoxified heat-labile toxin adjuvant dmLT. The authors explicitly left open whether parenteral SLA-SE can elicit mucosal functional antibodies in humans, whether the SLA-SE/dmLT synergy translates to human vaccination, whether serum functional antibodies reliably proxy mucosal functional immunity outside the mouse system, and why SLA-SE benefited the dmLT-driven response more than the CfaEB-driven response. No SLA-SE-containing ETEC vaccine has entered human trials, human parenteral ETEC vaccine development (CssBA+dmLT intramuscular; dscCfaE+LT(R192G) intradermal; dmLT intradermal) has proceeded without SLA-SE and without direct intestinal functional-antibody readouts, and both mechanistic hypotheses for the differential adjuvant benefit remain untested; the problem is therefore open.

Background

Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of diarrheal illness worldwide, especially among children in low- and middle-income countries and among travelers; no licensed ETEC vaccine exists. ETEC colonizes the small intestine via fimbrial colonization factors, of which colonization factor antigen I (CFA/I) is one of the most prevalent, and secretes heat-labile toxin (LT), an enterotoxin that drives secretory diarrhea. Protective immunity is believed to require functional antibodies at the intestinal surface — antibodies that block fimbriae-mediated adherence (measurable, for example, as inhibition of CFA/I-mediated hemagglutination, the HAI assay) or neutralize LT. CfaEB is a recombinant antigen comprising the major and minor (adhesive) subunits of CFA/I; dmLT (LT R192G/L211A) is a double-mutant, detoxified LT that retains adjuvant activity and is itself a vaccine antigen candidate. Because oral protein vaccines face gastric degradation and oral tolerance, and intranasal LT-adjuvanted vaccines caused Bell's palsy in trials, there is strong interest in achieving mucosal immunity by the injection (parenteral) route. TLR4 agonists are bacterially derived or synthetic lipid A analogs that stimulate the innate receptor TLR4 and are widely used vaccine adjuvants; SLA (a second-generation lipid A analog optimized from glucopyranosyl lipid A, GLA) formulated in a squalene-in-water stable emulsion (SLA-SE) is one such clinical-stage adjuvant platform, developed by the Infectious Disease Research Institute (now Access to Advanced Health Institute). In 2019, Liang and colleagues reported that, in mice, intramuscular immunization with CfaEB plus SLA-SE elicited serum and intestinal IgA and IgG responses at least as strong as dmLT, enhanced IgG2a (a mouse IgG subclass associated with T-helper-1-skewed immunity) and LT-neutralizing serum antibodies elicited by dmLT four- to eight-fold, and synergized with dmLT for several mucosal and systemic readouts. Notably, mucosal and serum functional (HAI) responses correlated strongly (Spearman 0.74-0.80), but only in the mouse system. The authors stated that further studies would be necessary to determine whether parenteral SLA-SE can elicit mucosal immunity in humans, that the SLA/dmLT synergy needed translation to human volunteers, and that the reason SLA-SE benefited the dmLT-driven response more than the CfaEB-driven response was unclear (speculating that the roughly tenfold-lower dmLT dose, or anti-dmLT antibodies clearing or neutralizing dmLT at boosting, might contribute). Related human work before and after: detoxified LT adjuvants (dmLT, LT(R192G)) have been given to humans by sublingual, oral, intradermal, and intramuscular routes with acceptable safety, and the related TLR4 agonist GLA-SE has completed multiple Phase 1/2 trials — but with systemic immunogenicity endpoints, so the capacity of a parenteral TLR4-agonist emulsion to program intestinal functional antibody immunity in humans remains empirically untested.

Problem Statement

Determine whether the mouse finding that parenteral (intramuscular) vaccination with a TLR4-agonist stable-emulsion adjuvant (SLA-SE) can program functional mucosal immunity translates to humans, and what biological mechanism explains the antigen-dependence of the effect. Concretely, the following linked questions — posed by Liang et al. (npj Vaccines 2019;4:19, DOI 10.1038/s41541-019-0116-6) — remain unresolved as of August 2026: (1) Can intramuscular immunization with an SLA-SE-adjuvanted vaccine antigen reproducibly elicit functional mucosal antibodies (for example, intestinal IgA or IgG that inhibits CFA/I-mediated hemagglutination or neutralizes LT) in humans? (2) Does the synergy observed in mice between SLA-SE and dmLT transfer to human vaccination? (3) Are serum functional antibody responses a reliable proxy for mucosal functional antibody immunity in humans, as they appeared to be in the mouse system (Spearman 0.74-0.80)? (4) By what mechanism did SLA-SE benefit the dmLT-driven response more than the CfaEB-driven response — in particular, does the roughly tenfold-lower dmLT dose, or rapid clearance/neutralization of dmLT by anti-dmLT antibodies induced at priming, account for the difference? These questions jointly serve one scientific objective — determining, in humans and mechanistically, whether the parenteral TLR4-agonist route can program functional mucosal immunity.

References

  1. Liang H, Poncet D, Seydoux E, Rintala ND, Maciel M, Ruiz S, Orr MT. 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. https://doi.org/10.1038/s41541-019-0116-6
  2. Poncet D, Hessler C, Liang H, Gautheron S, Sergent M, Rintala ND, Seydoux E, Huang PD, Argilla D, Ruiz S, Heinrichs J, Maciel M Jr, Orr MT. Preclinical optimization of an enterotoxigenic Escherichia coli adjuvanted subunit vaccine using response surface design of experiments. npj Vaccines 2020;5:83. DOI 10.1038/s41541-020-00228-w. https://doi.org/10.1038/s41541-020-00228-w
  3. Lee T, Gutiérrez RL, Maciel M, Poole S, Testa KJ, Trop S, Duplessis C, Lane A, Riddle MS, Hamer M, Alcala A, Prouty M, Maier N, Erdem R, Louis Bourgeois A, Porter CK. Safety and immunogenicity of intramuscularly administered CS6 subunit vaccine with a modified heat-labile enterotoxin from enterotoxigenic Escherichia coli. Vaccine 2021;39(39):5548-5556. DOI 10.1016/j.vaccine.2021.08.032. https://doi.org/10.1016/j.vaccine.2021.08.032
  4. Gutiérrez RL, Riddle MS, Porter CK, Maciel M Jr, Poole ST, Laird RM, Lane M, Turiansky GW, Jarell A, Savarino SJ. A first in human clinical trial assessing the safety and immunogenicity of two intradermally delivered enterotoxigenic Escherichia coli CFA/I fimbrial tip adhesin antigens with and without heat-labile enterotoxin with mutation LT(R192G). Microorganisms 2023;11(11):2689. DOI 10.3390/microorganisms11112689. https://doi.org/10.3390/microorganisms11112689
  5. Gutiérrez RL, Porter CK, Harro C, Talaat K, Riddle MS, DeNearing B, Brubaker J, Maciel M Jr, Laird RM, Poole S, Chakraborty S, Maier N, Sack DA, Savarino SJ. Efficacy evaluation of an intradermally delivered enterotoxigenic Escherichia coli CF antigen I fimbrial tip adhesin vaccine coadministered with heat-labile enterotoxin with LT(R192G) against experimental challenge with enterotoxigenic E. coli H10407 in healthy adult volunteers. Microorganisms 2024;12(2):288. DOI 10.3390/microorganisms12020288. https://doi.org/10.3390/microorganisms12020288
  6. Pasetti MF, Milletich PL, White JA, Butts J, Brady RC, Dickey MD, Ballou C, Maier N, Sztein MB, Baqar S, Louis Bourgeois A, Bernstein DI. Safety and immunogenicity in humans of enterotoxigenic Escherichia coli double mutant heat-labile toxin administered intradermally. npj Vaccines 2025;10(1):23. DOI 10.1038/s41541-025-01071-7. https://doi.org/10.1038/s41541-025-01071-7
  7. Nafade V, Sule NO, Zhang Y, Sassi A, Basta NE. Vaccine candidates designed to prevent enterotoxigenic Escherichia coli diarrhoea: a scoping review of clinical trials evaluating vaccine candidates in development. Tropical Medicine & International Health 2026;31(3):295-306. DOI 10.1111/tmi.70070. https://doi.org/10.1111/tmi.70070
  8. Crothers JW, Norton EB. Recent advances in enterotoxin vaccine adjuvants. Current Opinion in Immunology 2023;85:102398. DOI 10.1016/j.coi.2023.102398. https://doi.org/10.1016/j.coi.2023.102398
  9. Vickers TJ, Buckley DP, Khatoon N, Sheikh A, Setu B, Berndsen ZT, Fleckenstein JM. Parenteral vaccination with recombinant EtpA glycoprotein impairs enterotoxigenic E. coli colonization. Infection and Immunity 2025;93(6):e00601-24. DOI 10.1128/iai.00601-24. https://doi.org/10.1128/iai.00601-24
  10. Zhou J, Liu B, Yang Q, Zhou J, Zheng J, Chen Y, Fu L, Du J, Zhang Z, Zhang J, Li C. Licensed and investigational TLR4 agonists as vaccine adjuvants: structural basis, clinical progress, and future directions. Frontiers in Immunology 2026;17:1872018. DOI 10.3389/fimmu.2026.1872018. https://doi.org/10.3389/fimmu.2026.1872018
  11. Svennerholm AM, Lundgren A, Leach S, Akhtar M, Qadri F. Mucosal immune responses against an oral enterotoxigenic Escherichia coli vaccine evaluated in clinical trials. Journal of Infectious Diseases 2021;224(12 Suppl 2):S821-S828. DOI 10.1093/infdis/jiab475. https://doi.org/10.1093/infdis/jiab475
  12. Bernstein DI, Pasetti MF, Brady R, Buskirk AD, Wahid R, Dickey M, Cohen M, Baughman H, El-Khorazaty J, Maier N, Sztein MB, Baqar S, Bourgeois AL. A Phase 1 dose escalating study of double mutant heat-labile toxin LTR192G/L211A (dmLT) from Enterotoxigenic Escherichia coli (ETEC) by sublingual or oral immunization. Vaccine 2019;37(4):602-611. DOI 10.1016/j.vaccine.2018.12.011. https://doi.org/10.1016/j.vaccine.2018.12.011
  13. ClinicalTrials.gov NCT02071758: Phase 1 LEISH-F3 + SLA-SE Vaccine Trial in Healthy Adult Volunteers (sponsor: Access to Advanced Health Institute; completed 2015; no published results). https://clinicaltrials.gov/study/NCT02071758
  14. ClinicalTrials.gov NCT07237282: A Phase 1 Clinical Trial of Adjuvanted Protein-based HCV Vaccine Candidates (E1E2 heterodimer + SLA-SE; not yet recruiting as of August 2026). https://clinicaltrials.gov/study/NCT07237282