Purification and Study of CC Chemokine-Based Strategies to Combat Chronic Inflammation and HIV

The human immune system is among one of the most complex organ systems in the human body. While it is responsible for healing wounds and protecting from disease, it can also have deleterious effects when over- or under-active. For example, many people who experience traumatic brain injury (TBI) suffer many of their ill effects due to the increased pressure on their brain, caused by an influx of immune cells. In another example, Auto-Immune Deficiency Syndrome (AIDS) is a progression of the Human Immunodeficiency Virus (HIV) that results in the death of immune cells, and thus, leaves the person open to opportunistic infections. Proteins involved in both these immune processes are known as chemokines (chemoattractant cytokines), which are small, secreted proteins that recruit and activate leukocytes, and can signal their movement towards a site of inflammation or infection (known as chemotaxis). As a result, more detailed study of the chemokine system is necessary in order to discover novel strategies to combat immune system-related ailments. This dissertation focuses on the broad applications involving utilizing certain segments of the chemokine system as biotherapeutics in chronic inflammation and HIV infection. First, the purification and structural study of the poxvirus viral CC Chemokine Inhibitor (vCCI) that naturally inhibits chemotaxis helps shed light on the mechanism behind this protein’s broadly-applicable method of binding CC Chemokines, revealing key residues that are likely vital for the mechanism behind the protein’s ability to broadly inhibit inflammation. Later, CC Chemokines are also studied in the context of HIV-inhibition, focusing on the N-terminal properties of a natural chemokine analog, 5P12-RANTES, which has been shown to inhibit HIV entry into the human cell. This inhibitory chemokine analog is also used to design and create second-generation HIV entry inhibitors, 5P12-Linker-T1144 and 5P12-Linker-T2635, which inhibit HIV at two separate stages of HIV entry process. And finally, chemokine analogs are suggested as a possible system for targeting cells likely to contain latent HIV DNA, to later activate transcription and allow the HIV-infected cells to be recognized by the immune system by utilizing the protein 5P14-Linker-Tat. Overall, this dissertation reveals structural insights into the proteins vCCI, vMIP-II and 5P12-RANTES that help elucidate the properties of the chemokine system, as well as offers three newly-engineered proteins, 5P12-Linker-T1144, 5P12-Linker-T2635 and 5P14-Linker-Tat to be used to combat HIV infection with preliminary results showing signs of efficacy