Abstract

Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identified recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1β, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-specific fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune profile. Liposomes containing GLA and 3M-052 together elicited both LecA-specific fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modifications to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in significantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis.

Highlights

  • Amebiasis, a diarrheal disease caused by the enteric protozoan parasite Entamoeba histolytica, is responsible for ~50 million infections annually worldwide, causes traveler’s diarrhea, and disproportionately affects disadvantaged populations.[1,2] For example, cases of amebiasis disease in Mexico from 2000–2010 dramatically outnumbered the combined number of cases of dengue, tuberculosis, malaria, AIDS, leishmaniasis, Chagas disease, and multiple other infectious diseases.[3]

  • Formulations were analyzed by HPLC-CAD for Glucopyranosyl lipid adjuvant (GLA) content after consisted of dipalmitoyl phosphatidylcholine (DPPC), PEGylated manufacture and the GLA concentration was within 20% of the ParƟcle Diameter (Z-ave, nm) Size Polydispersity Index

  • After 12–16 months storage at 5 °C, the 3M-052 no change in particle size over 24 h after mixing with antigen content for all batches was within 20% of the initially measured whether stored at 5 °C or ambient temperature (Fig. S4), values (Fig. S3), the data should be interpreted with liposomes containing DSPE-PEG2000 showed a slightly greater caution since different HPLC methods were employed (see tendency to increase in size compared to the liposomes containing the shorter PEG length (DSPE-PEG750)

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Summary

Introduction

A diarrheal disease caused by the enteric protozoan parasite Entamoeba histolytica (a biodefense category B pathogen), is responsible for ~50 million infections annually worldwide, causes traveler’s diarrhea, and disproportionately affects disadvantaged populations.[1,2] For example, cases of amebiasis disease in Mexico from 2000–2010 dramatically outnumbered the combined number of cases of dengue, tuberculosis, malaria, AIDS, leishmaniasis, Chagas disease, and multiple other infectious diseases.[3]. Preschool children from a Bangladesh cohort showing E. histolytica-specific gut IgA and systemic IFN-γ response were protected from recurrent infections.[5,6]. E. histolytica infection starts with the ingestion of amebic cysts from contaminated food or water, which after excystation form trophozoites in the intestinal lumen. These adhere to epithelial cells through interaction of galactose and N-acetyl-Dgalactosamine (Gal/GalNAc)-specific lectin with host Gal/GalNAccontaining glycoconjugates. The trophozoites kill and ingest host cells in a lectin-dependent manner.[7,8] Blockade of lectin activity with Gal or GalNAc prevents contact-dependent cytolysis.[8]

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