Biology Project: Gene Expression Analysis

This project became my master's thesis while I worked at Plum Island Animal Disease Center. Below is the abstract; you can find the entire thesis along with other published work at my research gate profile (You can click on the beaker icon in the top right-hand corner to access my research gate profile).

Gene Expression Analysis in Bovine Peripheral Blood Mononuclear Cells in Response to Adenovirus-Vectored Foot-and-Mouth Disease Vaccine

Foot-and-mouth disease virus (FMDV) gives rise to a highly contagious and economically important disease of cloven-hooved animals. Vaccination reduces the economic impact by inducing serotype-specific protection. Recently, a replication-defective adenovirus-vectored foot-and-mouth disease (FMD) subunit vaccine was developed and licensed. Serum virus neutralization (SVN) titer ≥1.5 to FMDV is the best predictor of vaccine-induced protection. However, protection does not always correlate with the presence of neutralizing antibodies. For example, some animals with high SVN titer develop signs of disease, and conversely, some animals with negligible SVN titer are protected. Consequently, a gap exists in our understanding of vaccine-induced protection in cattle administered the replication-defective Ad-5 vectored FMDV subunit vaccine (Ad-5 FMDV). I hypothesized that gene expression analysis of bovine peripheral blood cells would provide a genomic tool for predicting Ad5-FMDV vaccine efficacy for foot-and-mouth disease. Thus, I sought to identify genes associated with protection after vaccination with Ad-5 FMDV. Microarray-based analysis of mRNA transcripts from peripheral blood leukocytes of vaccinated cattle (n=21), drawn on 0, 1, 14 and 15 days post-vaccination, revealed that in response to Ad-5 FMDV, protected cattle (n=5) demonstrated a rapid but short-lived induction of stress-related genes. Vaccination with the adenovirus vaccine vector alone, ad-null, (n=4) resulted in a similar, but less robust, expression pattern, whereas unprotected cattle (n=4) exhibited an initially mild, but increasing expression profile of stress-related genes. A subset of 12 immune response-related genes was verified by RT-qPCR. A separate list of genes that correlated with protection was identified. One gene in particular, CCL8, an inflammatory mediator, resulted in a transient up-regulation that was associated with protection, whereas vaccinated, but unprotected animals, exhibited a pattern of prolonged expression that did not return to baseline levels. Rapid but transient induction of the CCL8 gene in Ad5-FMDV vaccinated cattle correlated with protection, irrespective of SVN titer. These microarray-based results were verified with RT-qPCR using a TaqMan™ probe. Capture ELISA was unable to detect the CCL8 protein in plasma. This small study was expanded to a larger cohort of cattle (n=39) utilizing RT-qPCR. From the 32 protected cattle in this expanded study, we were unable to conclude that transient up-regulation of CCL8 gene following vaccination correlated with protection. Thus, although CCL8 transient up-regulation could not be confirmed as a sole correlate of immune protection, it may be possible to assign the transient up-regulation of CCL8 as a mechanistic immune function that contributes to protection with other mechanistic immune functions that supplement or co-correlate with protection. Perhaps transient up-regulation of CCL8 is one of multiple biomarkers that contribute to protection. Once the biological underpinnings of protection are better understood perhaps these complex correlates of vaccine-induced protection will be readily identified.

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