Transcutaneous Administration of Dengue Vaccines

Abstract

In the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (TC) route. Vaccines against type 2 Dengue virus particles (DENV2 New Guinea C (NGC) strain) combined with enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) were administered to BALB/c mice in a three-dose immunization regimen via the TC route. As a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (ID) route. Our results showed that mice vaccinated either via the TC or ID routes developed similar protective immunity, as measured after lethal challenges with the DENV2 NGC strain. Notably, the vaccine delivered through the TC route induced lower serum antibody (IgG) responses with regard to ID-immunized mice, particularly after the third dose. The protective immunity elicited in TC-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and IgG subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. Altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the TC route and offer perspectives for future clinical applications.

Keywords: adjuvant; dengue vaccines; heat-labile toxin; intradermic immunization; transcutaneous immunization.

Figure 1

Serum IgG responses elicited in mice immunized with type 2 Dengue virus particles (DENV2) through transcutaneous (TC) or intradermal (ID) administration routes. Concentrated virus particles (10 or 50 µg protein/dose) were administered alone or with heat-labile toxin (LT) as adjuvant (10 µg/dose) in BALB/c mice (n = 5). The immunization protocol consisted of 3 doses administered with a two week interval. Both TC as well as ID inoculations were administered in the dorsum of the animals, as described in the Material and Methods section. (A,B) Dose-dependent anti-DENV serum IgG responses elicited in mice following administration via TC (A) or ID (B) routes. Columns represent mean IgG concentration values + SD. (C,D) Individual serum anti-DENV2 IgG responses in mice submitted to TC (C) or ID (D) immunizations measured two weeks after the last dose. Statistical analysis was performed with a two-way ANOVA test associated with Bonferroni’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Representative results of 3 independent experiments.

Figure 2

Serum antibody responses against DENV2 induced after TC or ID immunizations. DENV-specific serum antibody responses induced after TC and ID 3rd immunization doses, measured by ELISA with concentrated DENV particles as solid phase (n = 5). (A,B) Serum IgG subclass responses in mice immunized via TC (A) or ID (B) routes. Values indicated above the lines represent IgG1/IgG2a ratio, measured in each immunized group. (C,D) Binding of anti-DENV2 antibodies from serum samples of immunized mice via TC (C) and ID (D) routes to intact and heat-denatured virus particles. (E,F) Antigen binding affinity of anti-DENV2 antibodies induced after immunization via TC (E) or ID (F) routes. Serum samples from mice submitted to TC (E) or ID (F) immunizations were tested by ELISA using different concentrations of ammonium thiocyanate as a dissociating agent. Statistical analyses were performed by two-way ANOVA in association with Bonferroni’s post-test * p < 0.05, **** p < 0.0001. Representative results of 2 independent experiments.

Figure 3

Cytokine secretion profile detected in mice submitted to TC and ID immunizations with DENV2 particles. Spleens of immunized mice (n = 5/ID 10 µg + LT n = 4) were harvested two weeks after the 3rd immunization dose and cytokines secreted by in vitro cultivated cells were determined according to procedures described in the Material and Methods section. (A–C) Th1 profile cytokines: interferon gamma (IFN-γ) (A), tumor necrosis factor alpha (TNF-α) (B) and interleukin 2 (IL-2) (C). (D,E) Th2 profile cytokines: IL-4 (D) and IL-10 (E). Statistical analysis was performed by two-way ANOVA in association with Bonferroni’s post-test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Protective immunity induced in mice after immunization via TC or ID routes with DENV2 New Guinea C (NGC). Survival and morbidity curves were mounted after inoculation of lethal intracranial (i.c.) challenge with 1 × 10⁶ NGC virus particles 2 weeks after the 3rd immunization dose (n = 5). (A,B) Survival curves of mice immunized via TC (A) or ID (B) routes following a lethal challenge with the DENV2 NGC strain. (C,D) Morbidity curves of mice immunized via TC (C) or ID (D) routes following the lethal challenge. Statistical analysis performed by Mantel–Cox test. * p < 0.05.