Knowledge of the interactions between Giardia Lamblia and its intestinal milieu is extremely limited. The hypothesis underlying this project is that the great variations in clinical manifestations and duration of giardiasis are due to parasite interactions with both immune and non- immune components of this environment. The long-term objective of these investigations is to gain valuable basic information on the aspects of parasites of parasite biology and host defenses that determine colonization and persistence of trophozoites in the human small intestine. The first Specific aim is to study the human secretory antibody response in depth, focusing upon the isotype and antigenic specificity of antibodies that inhibit trophozoite motility or attachment, and, conversely, the effects of intestinal conditions on antigen expression, interactions with antibody and trophozoite evasion of secretory defenses. These studies should provide important new insights into the variable severity of giardiasis. To determine how the surface of trophozoites adapts them to survive in the small intestine, we have cloned, expressed in E. coli and sequenced an important trophozoite surface antigen gene (called 417). Antibodies against the recombinant protein inhibit trophozoite attachment and growth and immunoprecipitate trophozoite surface proteins of -66 and 85 kD. Therefore, in Specific Aim 2, we will compare NH2-terminal amino acid sequences and peptide maps of purified p66 and p85 to determine their relationship to each other and to the 417 gene, and ask whether and how they are post-translationally processed or covalently modified and which epitopes are exposed to the host environment. Concomitantly, in Specific Aim 3, we will ask whether inhibition of attachment by antibody to p417 is due to agglutination. Next, we will assess the degree of cross-linking a native p66 and p85 on the trophozoite surface, as well as their interactions with intestinal proteases, bile salts, and mucins, and determine whether expression of p66 and p85 is regulated by intestinal stimuli.
In Specific Aim 4, we will immunize animals orally with p417 and determine whether the secretory antibody response is protective or whether trophozoites can effectively evade it. These studies should provide critical new insights into the biologic and immunologic interactions of an important pathogen with the complex intestinal environment.
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