The Pharmacodynamic Functions of Low-Dose Rapamycin as a Model for Universal Influenza Protection

Gust, William (2017) The Pharmacodynamic Functions of Low-Dose Rapamycin as a Model for Universal Influenza Protection. Undergraduate thesis, under the direction of John Rimoldi from BioMolecular Sciences, University of Mississippi.

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Abstract

One of the most sought after breakthroughs in modern influenza research is the creation of a universal influenza vaccine that would protect against all strains of avian influenza. An unlikely model for this kind of protection might be found in the mTOR inhibitor rapamycin. Traditionally used to prevent organ transplant rejection, low doses of rapamycin have been found to protect mice against infection with distinct subtypes of influenza. However, it is not understood how a partial block in this pathway provides optimal heterosubtypic immunity. Interestingly, previous experiments have shown that low dose rapamycin inhibits germinal center formation to reduce antibody class switching and generate cross-protective influenza antibodies. Yet, it is not known how these antibodies, which do not neutralize the virus, enhance protection against distinct influenza subtypes. The focus of my research was to further characterize the antibody protection and mTORC1 downstream effector regulation mediated by low dose rapamycin. Significant mechanisms of viral clearance by non-neutralizing antibodies include complement-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). To investigate whether these mechanisms are required for rapamycin-mediated antibody protection in vivo, we inhibited the complement system in vivo using Cobra Venom Factor (CVF), depleted natural killer cells in vivo using an anti-NK1.1 antibody, and compared the efficacy of rapamycin treatment between control and depleted mice following H5N1 infection in both cases. We also performed an in vitro ADCC assay with serum from PBS or rapamycin-treated mice. Based on our data, we concluded that rapamycin-mediated antibodies do not use the complement system or ADCC to provide heterosubtypic influenza protection. To understand how low-dose rapamycin specifically alters the expression of certain downstream mTOR effectors, we ran quantitative Polymerase Chain Reaction (qPCR) analyses on RNA isolated from PBS and rapamycin-treated B cells. Additionally, we ran western blots on PBS and rapamycin-treated B cells to investigate how low-dose rapamycin uses phosphoregulation to alter downstream mTORC1 protein expression. Based on our data, we found that rapamycin decreases expression of the Slc2a1 gene, which encodes Glucose transporter 1 (GLUT1), and the phosphoprotein pS6, which helps encode B cell ribosome biogenesis. In addition, rapamycin may alter the expression of other genes, but to a lesser extent.

Item Type: Thesis (Undergraduate)
Creators: Gust, William
Student's Degree Program(s): B.S. Pharmaceutical Sciences
Thesis Advisor: John Rimoldi
Thesis Advisor's Department: BioMolecular Sciences
Institution: University of Mississippi
Subjects: Q Science > Q Science (General)
Depositing User: Mr. William Gust
Date Deposited: 22 May 2017 13:12
Last Modified: 22 May 2017 13:12
URI: http://thesis.honors.olemiss.edu/id/eprint/957

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