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Authors:To, Albert
Contributors:Lehrer, Axel T. (advisor)
Biomed Science (Tropical Medicine) (department)
Insect-cell Expression
show 4 moreMice
Protein Purification
Protein Subunit Vaccine
Viral Glycoprotein
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Date Issued:2020
Publisher:University of Hawai'i at Manoa
Abstract:The ability to rapidly produce industrial quantities of antigen, for use in vaccinations, diagnostic tests and serosurveillance etc., is an essential component of an effective public health strategy. Properly-folded and post-translationally modified recombinant viral glycoproteins are important immunogens, especially used for the containment of highly virulent pathogens, however large scale production for preventative and therapuetic use are bottle-necked by the low protein yields and high cost associated with mammlian cell culture. The Drosophila S2 insect cell expression system is an ideal, alternative platform for expressing native-like proteins as it is capable of eukaryotic post-translational modification, requires lower maintenance than mammalian cells and is easily scalable. Furthermore purification using immunochromatography eliminates the need for peptide tags and yields protein at a high purity.
To generate our vaccine antigens, we have developed a production and purification method for the surface glycoproteins of the Sudan Ebolavirus (SUDV), Zika Virus (ZIKV), and Lassa Virus (LASV), expressed in stably transformed Drosophila S2 cell lines and purified using immunochromatography with an appropriate monoclonal antibody. Each of these glycoproteins was recognized by well-characterized antibodies, or convalescent sera from infected animals. When formulated with pre-clinical and clinical grade adjuvants, each of these surface glycoproteins generated maximal antibody titers in vaccinated mice after the second dose that only increased slightly after a third dose. We demonstrate that these glycoproteins can cross-react with glycoproteins from closely related viruses but do not exert any immunointerference. Protection from viral challenge was observed in mice receiving two doses of our ZIKV vaccine candidate, or a passive transfer of high ZIKV E-specific IgG titered sera from vaccine-immunized mice. Formulation of LASV GP with several adjuvants generated a diversified IgG response and a robust cell-mediated response with one of the tested formulations.
Together these results suggest that S2-cell expressed vaccine antigens, purified using immunoaffinity chromatography, are native-like in their conformational folding and can be used as effective immunogens when formulated with the appropriate adjuvant, capable of protecting mice from the targeted viral infection. Additionally, the work presented in this dissertation validates the use of insect cell-expressed viral glycoproteins in vaccines and will provide a cheaper and safer alternative than traditional vaccine platforms and can provide greater coverage, even to the vulnerable populations. This is especially important when rapid antigen production is required in response to a public health emergency such as the ongoing COVID-19 pandemic.
Pages/Duration:149 pages
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: Ph.D. - Biomedical Sciences (Tropical Medicine)

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