Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

David S. Bradley

Abstract

Passive antibody treatments are used to target infectious disease, toxins, venoms, and cancer antigens. Recently, there has been an increased interest in the use of avian-derived antibody treatments such as IgY. IgY is the primary serum antibody isotype present in the avian system, and IgY treatments have already been demonstrated to be effective against a variety of bacterial and viral infectious agents. There are two forms of IgY expressed in anseriformes birds, a full length IgY that is functionally similar to mammalian IgG, and an alternatively spliced IgY, IgY(ÄFc), that is comparable to the mammalian F(ab')2 fragment. The difference in structure between IgY and mammalian IgG, prevents IgY from interacting with mammalian Fc receptors, complement, and other inflammatory factors. The phylogenetic distance between mammalian and avian species allows IgY to have a higher avidity for certain mammalian epitopes and a unique antibody repertoire is developed compared to mammals, further enhancing the therapeutic potential of IgY.

Our current research is focused on developing goose IgY anti-viral treatments and ensuring the safety of these treatments in humans. The viral antigens of focus in this research are dengue virus type 2 (DENV-2) and the Andes virus (ANDV). In an attempt to generate monoclonal goose IgY antibodies, using modified mammalian hybridoma techniques, geese were immunized with DENV-2 antigen and blood was collected as a source of immune B-cells and fused with mammalian myeloma cells. Short lasting, virus- specific IgY producing hyrbidomas were created. To generate ANDV specific goose IgY antibodies geese were vaccinated with a DNA vaccine PWRG/AND-M, containing the full-length M genome segment of ANDV, via a needle-free device at two week intervals up to eight weeks and then at 12 weeks. One year later the same geese were booster vaccinated with either pWRG/AND(opt) or pWRG/AND(opt2) six times over a ten week time period. Average neutralizing titers of sera collected from geese six weeks after the primary vaccination was 10,000. Titers remained at this level for the one year in between vaccinations and then increased to nearly 100,000 after booster vaccination. Epitope mapping confirmed the specificity of the goose- derived antibodies and identified unique highly reactive epitopes. IgY from the initial vaccination recognized 11 epitopes across the M segment, and an additional 9 epitopes after booster vaccination. In vivo survival studies in a lethal challenge model of ANDV infection established the post-exposure treatment potential of the ANDV specific IgY.

To test the safety of the anti-viral IgY treatments for use in humans in vivo and in vitro safety experiments were completed. In a single injection study, mice were injected with a single dose of IgY/IgY(ÄFc) or PBS, and in a multiple injection study, rabbits were injected with multiple doses of IgY/IgY(ÄFc), IgY(ÄFc), human immunoglobulin, or PBS. Organs were collected after injection, hematoxylin and eosin stained, and scored by a blinded pathologist for abnormal pathology and/or inflammation. There were no inflammatory manifestations in the organs from animals in either the single or multiple injection study receiving IgY/IgY(ÄFc) or IgY(ÄFc). PBMCs and neutrophils were isolated from fresh human blood and co-cultured with IgY/IgY(ÄFc), mammalian IgG, and other controls. Culture supernatants were collected at various time points and analyzed for the presence of IL-1â, TNF-á, IL-10, neutrophil elastase, and nitric oxide using kit-based assays. All assays reported less reactivity of goose IgY/IgY(ÄFc) with human PBMCs and neutrophils compared to mammalian IgG and positive control mitogens. These results further support the lack of reactivity of avian IgY in the mammalian system and the benefits of safely using IgY as a treatment in the mammalian system.

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