Conserved metabolic enzymes as vaccine antigens for giardiasis.

Giardia lamblia is a leading protozoal cause of diarrheal disease worldwide. Infection is associated with abdominal pain, malabsorption and weight loss, and protracted post-infectious syndromes. A human vaccine is not available against G. lamblia. Prior studies with human and murine immune sera have identified several parasite antigens, including surface proteins and metabolic enzymes with intracellular functions. While surface proteins have demonstrated vaccine potential, they can exhibit significant variation between G. lamblia strains. By comparison, metabolic enzymes show greater conservation but their vaccine potential has not been established. To determine whether such proteins can serve as vaccine candidates, we focused on two enzymes, α-enolase (ENO) and ornithine carbamoyl transferase (OCT), which are involved in glycolysis and arginine metabolism, respectively. We show in a cohort of patients with confirmed giardiasis that both enzymes are immunogenic. Intranasal immunization with either enzyme antigen in mice induced strong systemic IgG1 and IgG2b responses and modest mucosal IgA responses, and a marked 100- to 1,000-fold reduction in peak trophozoite load upon oral G. lamblia challenge. ENO immunization also reduced the extent and duration of cyst excretion. Examination of 44 cytokines showed only minimal intestinal changes in immunized mice, although a modest increase of CCL22 was observed in ENO-immunized mice. Spectral flow cytometry revealed increased numbers and activation state of CD4 T cells in the small intestine and an increase in α4ß7-expressing CD4 T cells in mesenteric lymph nodes of ENO-immunized mice. Consistent with a key role of CD4 T cells, immunization of CD4-deficient and Rag-2 deficient mice failed to induce protection, whereas mice lacking IgA were fully protected by immunization, indicating that immunity was CD4 T cell-dependent but IgA-independent. These results demonstrate that conserved metabolic enzymes can be effective vaccine antigens for protection against G. lamblia infection, thereby expanding the repertoire of candidate antigens beyond primary surface proteins.

Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia.

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

Giardia Infection of the Small Intestine Induces Chronic Colitis in Genetically Susceptible Hosts.

The lumen-dwelling protozoan Giardia is an important parasitic cause of diarrheal disease worldwide. Infection can persist over extended periods with minimal intestinal inflammation, suggesting that Giardia may attenuate host responses to ensure its survival, although clearance eventually occurs in most cases. IL-10 is an anti-inflammatory regulator critical for intestinal homeostasis and controlling host responses to bacterial exposure, yet its potential role in coordinating antiprotozoal host defense in the intestine is not known. In this study, we found that murine infection with the natural enteric pathogen Giardia muris induced a transient IL-10 response after 2-4 wk at the primary site of infection in the upper small intestine, but parasite colonization and eradication were not affected by the absence of the cytokine in gene-targeted mice. However, IL-10 was critical for controlling infection-associated immunological sequelae in the colon because severe and persistent diarrhea and colitis were observed in IL-10-deficient mice within 1-2 wk postinfection but not in uninfected littermate controls. Inflammation was characterized by epithelial hyperplasia, neutrophil and macrophage expansion, and Th1 induction and could be prevented by blockade of IL-12/IL-23 p40 but not depletion of CD11c+ dendritic cells. Furthermore, the intestinal microbiota underwent characteristic shifts in composition and was required for disease because antibiotics and loss of TLR signaling in MyD88-deficient mice protected against colitis. Together, our data suggest that transient infection by a luminal and seemingly noninflammatory pathogen can trigger sustained colitis in genetically susceptible hosts, which has broader implications for understanding postinfectious syndromes and other chronic intestinal inflammatory conditions.