Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica

Abstract

Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired.

Keywords: Amebiasis; Entamoeba; Immune response; Liposome; Vaccine.

Fig. 1

Comparison of plasma IgG titers. Five mice per group were immunized three times subcutaneously at a 2-week interval with LecA antigen mixed with the corresponding adjuvant. Plasma samples collected a week after the final immunization were diluted 256,000-fold and analyzed for LecA specific IgG1 (black) and IgG2a (red) levels by ELISA. GLA-alum without LecA served as a control group. *IgG2a level elicited by these adjuvants were comparable and statistically significant (p < 0.05) in comparison with all other groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

LecA specific cytokine response. Mice were immunized subcutaneously at a two week interval as described. Splenocytes were isolated a week after third immunization and re-stimulated with LecA for 72 h. Production of extracellular IFN- γ, IL-17A, IL-2 and IL-4 were detected in the culture supernatants by Luminex. Black and orange circles denote cytokine levels upon re-stimulation either with medium alone or LecA respectively. * = p < 0.01; n.s. = not significant; RA = all-trans retinoic acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Prechallenge stool IgA response and adherence inhibitory potential. Mice were immunized with GLA 3M-052 liposome adjuvanted LecA using a mixed intranasal (weeks 0 and 4) and subcutaneous (week 2) regimen. Mice in the indicated groups received a weekly intraperitoneal injection of 150 μg RA. Stool samples were collected three weeks post third immunization. (A) Fecal supernatants were diluted 120-fold and prechallenge anti-LecA IgA titer was determined by ELISA; (B) potential of fecal IgA to inhibit adherence of trophozoites to mammalian cells was determined in vitro using adherence inhibition assay as described. * = p < 0.01; ** = p < 0.001; *** = p < 0.0001.

Fig. 4

Vaccine mediated protection in a mouse model of intestinal amebiasis. Mice immunized using a mixed regimen as described were challenged intracecally with E. histolytica four weeks post-final immunization. Mice were euthanized a week after the challenge and cecal contents analyzed for (A) antigen load using ELISA and (B) live ameba by culture as a measure of sterile immunity. Number of infected mice from the total challenged are indicated above each column. The efficacies for (adjuvant + LecA) and (adjuvant + LecA + RA) groups were calculated with regard to the corresponding control groups as described. Data from two independent but identical trials was pooled. * = p < 0.05.