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

Abstract

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.

Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin–Heat-Labile Toxin Chimera

“…While we speculate that a larger dose of the chimera antigen could possibly elicit comparable protection in NHP in the absence of the adjuvant, our data suggest that LT-derived adjuvants, such as LT(R192G), are able to enhance mucosal responses even if administered by non-mucosal routes. The final ETEC multivalent vaccine formulation will most likely require an adjuvant, and while the reactogenicity of the LT(R192G) is acceptable, as seen here and elsewhere (18), and its adjuvanticity has successfully on November 4, 2020 by guest http://iai.asm.org/ Downloaded from been evaluated in clinical trials by the transcutaneous (<clinicaltrials.gov>, #NCT01382095) and ID routes (#NCT01644565) (manuscripts in preparation), it is likely that future ETEC vaccine formulations would benefit from a less reactogenic LT toxoid. The recently developed LT(R192G/L211A) adjuvant (double mutant LT) (33) incorporates a second mutation that reduces reactogenicity, while preserving its adjuvanticity, when administered ID (18), and as such is a promising alternative.…”

“…The final ETEC multivalent vaccine formulation will most likely require an adjuvant, and while the reactogenicity of the LT(R192G) is acceptable, as seen here and elsewhere (18), and its adjuvanticity has successfully on November 4, 2020 by guest http://iai.asm.org/ Downloaded from been evaluated in clinical trials by the transcutaneous (<clinicaltrials.gov>, #NCT01382095) and ID routes (#NCT01644565) (manuscripts in preparation), it is likely that future ETEC vaccine formulations would benefit from a less reactogenic LT toxoid. The recently developed LT(R192G/L211A) adjuvant (double mutant LT) (33) incorporates a second mutation that reduces reactogenicity, while preserving its adjuvanticity, when administered ID (18), and as such is a promising alternative. The lack of an adjuvant-only group in the current NHP study precludes the assessment of the protection conferred by the anti-toxin-only response.…”

“…Importantly, immunization with chimera by both routes leads to the generation of high levels of functional antibodies, as judged by HAI assays, an in vitro proxy parameter for intestinal adhesion inhibition. Previous studies have demonstrated that, while dsc 19 CfaE is highly immunogenic when administered via different immunization routes, higher anti-adhesin and functional antibody levels were elicited with the co-administration of LT-or CT-based adjuvants (12, 13,18). In addition to adjuvant properties, those proteins also elicit anti-toxin responses that are important for protection, particularly against LT-producing (i.e.…”

“…We also observed modest anti-LTB IgG antibody titers (3.9 ± 0.38, mean ± SD) in the animals immunized ID with dsc 19 CfaE only, which was likely result of a minute contamination with LT(R192G) during dose preparation. In fact, we recently demonstrated that a dose as low as 0.01 µg of LT(R192G) is able to elicit an immune response (18). Mice given chimera ID had significantly greater anti-CfaE IgG titers than those given dsc 19 CfaE alone (P<0.001; Fig.…”

Preclinical optimization of an enterotoxigenic Escherichia coli adjuvanted subunit vaccine using response surface design of experiments

“…One of the first steps in the DoE approach is to define the range of doses that will be varied during the experiment. The upper bound of CfaEB was set at 10 µg as it was determined that two intradermal immunizations, 3 weeks apart, with 10 µg CfaEB (plus LT(R192G) or LT(R192G/L211A)) in BALB/c mice, reached a plateau in term of serum antigen-specific IgG and immunoglobulin A responses, as well as functional antibodies 53 . The upper bound of the SLA dose was fixed at 5 µg with an optimal dose comprised between 1 and 5 µg depending on both the overall vaccine formulation and the targeted immune response.…”