Dissertation/Thesis Abstract

Influence of Epitope Specificity on Antibody-mediated Complement Activation
by Chiampi, Alana, M.S., California State University, Long Beach, 2018, 47; 10830218
Abstract (Summary)

Candida albicans is a yeast-like fungal pathogen that in recent years has emerged as a frequently isolated, causative species of blood stream infections. Current anti-Candida treatments are limited and some can cause serious adverse side-effects. Furthermore, drug resistance in C. albicans has been on the rise. Consequently, there has been a push to develop new anti-Candida therapeutics. Our lab has been developing human recombinant antibodies as a potential therapeutic agent. These antibodies target different epitopes in mannan or mannose polysaccharides that are prominently displayed on C. albicans cells. One full-length anti-mannan antibody, M1g1, has been found to activate the alternative and classical complement pathways, as well as increase phagocytosis of C. albicans by human neutrophils, and increase the survival rate of mice treated with a lethal dose of C. albicans. A second full-length anti-mannan antibody, M2g1, was also constructed but stable cell lines for M2g1 production were not obtained. Both M1g1 and M2g1 share the same Fc region of IgG1, but they differ in the epitope binding region.

Previous studies have found that epitope specificity influences antibody biological functioning. The purpose of this study was to obtain a stable cell line to produce M2g1 antibody and study how the binding specificity of M2g1 influences its ability to activate complement. A variety of reagents were used to transfect the gene for M2g1 to Chinese hamster ovarian cells, and a non-liposomal reagent that forms positively charged complexes around negatively charged DNA produced stable transfectants for M2g1 production. M2g1 was purified with affinity chromatography. Flow cytometry and immunofluorescence microscopy were utilized to compare M2g1 and M1g1 at different amounts from 2.5 to 20µg/ml for their ability to bind to C. albicans cells and to activate C3 deposition. Binding to C. albicans by M2g1 was dose-dependent whereas binding by M1g1 reached saturation at lower amounts. At 20µg/ml, approximately 10% of cells treated with M2g1 showed fluorescence intensities above the background level, compared to 97% with M1g1. Deposition of C3 onto C. albicans was also much less in the presence of M2g1 than M1g1 at the amounts shown above. At 20µg/ml, approximately 30% of cells treated with M2g1 showed positive C3 binding, compared to 99% with M1g1. Furthermore, M1g1 binding to C. albicans and facilitation of C3b deposition was not enhanced or inhibited by M2g1 binding. These studies indicate that the epitope specificity of an antibody influences its biological functions. They also suggest that M2g1 may have a limited potential as an anti- Candida therapeutic.

Indexing (document details)
Advisor: Zhang, Mason
Commitee: Fraser, Deborah, Itatani, Carol
School: California State University, Long Beach
Department: Biological Sciences
School Location: United States -- California
Source: MAI 58/02M(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Microbiology
Keywords:
Publication Number: 10830218
ISBN: 978-0-438-56817-4
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