Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

David S. Bradley

Abstract

IgY, the predominate avian antibody isotype, has biochemical properties that make it attractive for a human immunotherapy: IgY does not bind to mammalian Fc receptor (FcR) or rheumatoid factor; IgY does not activate the mammalian complement; and IgY has no heteroagglutinins. Anseriformes (waterfowl, e.g. ducks and geese) produce 2 isoforms of IgY, full length IgY and alternatively spliced IgY lacking the Fc region and a stable equivalent to mammalian F(ab')2 fragment, and the predominate isoform following hyperimmunization. We, and others, have demonstrated that egg-derived avian polyclonal antibodies are prophylactic or therapeutic for a variety of different infectious agents including bacteria, viruses, and parasites.

Several routes of administration have been utilized for IgY, although the most intriguing has been those that are administered orally. The targets for the successful oral administrations have been associated with the gastrointestinal tract, other organ systems, and systemic infections. However, bioavailability of orally administered IgY has not been determined. As part of the research presented here, we administered purified goose-derived IgY via oral gavage to mice and determined seroconversion. Oral IgY is bioavailable and can be detected in the serum by 24 hrs. Multiple dosing and buffering to pH 8.0 resulted in higher serum titers, with the buffered IgY preparations not detected until 48 hrs. IgY was detectable up to 7 days post oral administration. Goose-derived IgY was relatively resistant to intestinal trypsin and chymotrypsin digestion but sensitive to gastric pepsin digestion, as demonstrated by others with IgY from other avian sources. This demonstration that orally administered IgY is bioavailable significantly increases the potential applications of IgY therapy. Furthermore the ability to administer IgY orally versus injection provides a novel and efficient means to treat disease worldwide.

In addition to determining the bioavailability of goose-derived IgY, we tested its therapeutic potential in two unrelated disease models. One organism of interest for the development of therapeutic IgY is dengue virus (DENV). At present, there are no anti-viral agents or vaccines approved to treat dengue-induced disease. Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), both disease manifestations originating from dengue virus infection are severe and life threatening. These disease states are mediated by serotype cross-reactive antibodies that facilitate antibody dependent enhancement (ADE) by binding to viral antigens and then Fc gamma receptors on additional myeloid cells. In this study we hypothesized that avian IgY, which does not interact with mammalian FcR, would provide a novel therapy for DENV. Polyvalent anti-DENV2 IgY was purified from eggs of DENV2-immunized geese and tested for its ability to neutralize and enhance a DENV2 infection both in vitro and in vivo. Our data suggests that DENV2 IgY is able to effectively neutralize DENV2 in the absence of inducing ADE. DENV2-specific epitopes were determined in both the full length and alternatively spliced goose IgY populations and were used to develop affinity purified DENV2 epitope specific antibodies.

The second organism used to test the therapeutic potential of IgY was Plasmodium berghei, the causative agent of murine cerebral malaria. Human cerebral malaria is a severe manifestation resulting from the infection of Plasmodium falciparum. Although there are successful antimalarial drugs on the market, there are increased reports of isolated strains that are drug resistant, therefore highlighting the need for new drug molecules or therapies to be used in combination therapy. Here we generated goose-derived IgY that was specific for Plasmodium berghei, or the merozoite surface protein 1 (MSP-1) antigens, and determined their ability to treat cerebral malaria in a murine model. Mice survived significantly longer and had decreased parasitemia when both malaria specific IgY were administered together on days 2 and 4. These data suggest that malaria specific IgY is a potential therapeutic candidate to be used in combination therapy in order to prolong death and provide time for the combination drug to be effective.

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