Eotaxin belongs to the chemokine family of proteins, which are involved in inflammation and in the development of the immune system, regulating activation and chemotaxis of leukocytes. Chemokines are involved in a variety of inflammatory diseases such as rheumatoid arthritis and allergic asthma. The viral protein CC-chemokine inhibitor (vCCI) from poxviruses can bind many different chemokines with high affinity, which makes it a potentially valuable therapeutic agent to treat such inflammatory diseases. However, it is not clear how vCCI is able to bind so many different chemokines. NMR data from the vCCI:MIP-1β complex and preliminary vCCI:eotaxin NMR data indicates that in eotaxin Phe11, Arg16, Arg 22, Lys 44, and possibly Lys 47 are involved in binding vCCI. Correspondingly, Tyr217, Phe215, Val185, along with Asp141, Glu143 and a flexible acidic loop in vCCI are involved in binding eotaxin. Mutations were made in these residues and binding to vCCI was tested by fluorescence anisotropy. This project investigates the interaction of chemokines and vCCI at a molecular level through NMR, mutagenesis, and fluorescence anisotropy studies. The results of investigation show that residues Phe11, Arg16, Arg 22, Lys 44 in eotaxin are involved in binding vCCI. Single mutations in Asp141, Glu143 of vCCI do not show significant change in binding affinity. More mutations such as a double mutant D141A/E143A vCCI, mutants of other nearby acidic residues (e.g. Asp73, Asp75), need to be done before any conclusion is made.

The global pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has underscored the urgent need for effective therapeutic strategies to inhibit viral replication and transmission. Although the severity of Coronavirus disease 2019 (Covid-19) has diminished, the SARS-CoV-2 virus is still circulating and evolving, meaning that there is still a distinct risk that a potent Variant of Concern (VOC) could arise at any time. This thesis summarizes our efforts in investigating the efficacy of several prophylactic treatments that were being investigated by the scientific community, and subsequently describes our efforts in probing the molecular mechanisms governing SARS-CoV-2 infection to identify novel vulnerabilities to exploit against the virus.The first portion of this dissertation orients the reader in understanding the origins and features of SARS-CoV-2 and coronaviruses in general. We then move into briefly presenting key structural features of the SARS-CoV-2 virion before introducing the Spike protein and its role in facilitating infection. We cover these topics with a focus on the structural aspects of the Spike protein, including its glycosylation pattern as well as its conformational changes as it engages the human Angiotensin Converting Enzyme 2 (hACE2) receptor. We finish by describing how SARS-CoV-2 virus can interact with proteins on the surface of cells to modulate its ability to infect different kinds of cells, both in vivo and in vitro. The second phase of the thesis focuses on summarizing our work in searching for overlooked epitopes for the development of antiviral prophylactics. Specifically, we describe our work in assessing the role of SARS-CoV-2 Spike S1 domain N-linked glycans in retaining viral infectivity and mediating viral entry into susceptible cells. In addition, we assess whether certain glycans are important in mediating viral capture and presentation by the C-type lectin receptor DC-SIGN. To quantify DC-SIGN capture, we developed a cell novel cell assay whereby DC-SIGN would deliver virions to susceptible cells, thereby facilitating infection-in-trans (referred to as Trans-infection in this document). Overall, our studies indicated that the glycans at N17 and N122 were important in retaining SARS-CoV-2 pseudotyped viral infectivity. On the other hand, removal of the glycan at N234 appeared to enhance viral infectivity. Drawing from insights gained in performing these assays, we then identified and tested clusters of glycans to ascertain if they were able to act as viable “cold spots” for the design and development of viral entry inhibitors. We conclude by assessing whether SARS-CoV-2 Spike protein from different strains altered the ability of Spike pseudotyped virions in infecting cells and in being captured by DC-SIGN. We then move into assessing the efficacy of the broadly-antiviral lectin Griffithsin against SARS-CoV-2 Spike pseudotyped viruses. We tested the importance of several variables that were relevant in the context of Covid-19, including the role of the additional SARS-CoV-2 structural proteins in mediating DC-SIGN recognition, the efficacy of Griffithsin against pseudoviral strains that expressed additional structural proteins, and the differences in inhibitory capability of several Griffithsin variants. We finish by describing several additional attempted assays that made use of our newfound expertise in SARS-CoV-2 pseudoviral production. This included several collaborations, ranging from assessing the extent to which gaseous ozone could sterilize SARS-CoV-2 pseudovirus-contaminated fomites, to troubleshooting the parameters to attempt to detect pseoudotyped lentiviral binding to functionalized Biolayer Interferometry (BLI) probes. Ultimately, this thesis contributes to the growing body of knowledge surrounding SARS-CoV-2 inhibition and prophylactic treatment by elucidating vulnerabilities in critical viral infection processes. These findings serve as the first steps to possibly develop novel prophylactic inhibitors that mitigate Covid-19 disease progression.

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.

Viruses have evolved mechanisms to subvert the immune system by interfering with chemokine signaling. The two mechanisms of evasion discussed in this thesis are proteins which bind chemokines to prevent them binding their receptors (as vCCI does) and chemokine analogs (such as vMIP-II) that competitively inhibit chemokines. Molecular dynamics simulations suggested that Y80 in vCCI is important for propping open the acidic loop, which keeps the binding site open. We investigated this hypothesis using ITC, NMR, and CD. For vCCI WT and vCCI Y80A, results included: a higher KD for the mutant (2.99 x 10-8 M) when binding vMIP-II compared to vCCI WT (≤1.00 x 10-10 M), key residues involved in binding shifted in the unbound mutant NMR HSQC spectrum, and the mutant had lower stability demonstrated by a 6.1°C reduction in Tm. The Y80A mutation lowers chemokine affinity, which may indeed be due to collapse of the substrate-binding loop.

Inflammation occurs when chemokines are secreted at a site of injury or infection, mediating a buildup of immune cells. Therefore, inhibiting chemokines in some cases could be an appealing target to control inflammation. Many viruses have evolved strategies to subvert the human chemokine system by producing chemokine binding proteins. Among them, poxviruses encode vCCI (viral CC chemokine inhibitor), which has been shown to bind more than 20 CC chemokines with high affinity. Therefore, understanding the mechanism of the high binding affinity between vCCI and CC chemokines could pave a way to develop vCCI as a new therapeutic candidate to treat inflammation related disease. In collaboration with the Prof. Michael Colvin group, combining an experimental approach and computational methodology, to unveil the possible key residues in the function of interaction between vCCI and chemokines, our work shows vCCI Y80, E143, and I184 residues are important for its binding to a chemokine vMIP-II (vCCL2). The vCCI mutation Y80R showed a loss of binding of 55 fold compared to the wild type of protein in BLI experiments, while E143K, I184R mutants decreased binding affinity of 37 and 25 fold respectively. We also found some key residues (G17, V44, Q45) of TARC (CCL17) involved in the interaction with vCCI. Highest affinity was achieved with the triple mutant CCL17 G17R/V44K/Q45R, which gave a Kd of 0.25 ± 0.13 µM for the vCCI:CCL17 variant complex, a 68 fold improvement in affinity compared to the complex with wild type CCL17. Silk fibroin (SF or silk in abbreviation) derived from the species Bombyx mori has been recognized as a material for biomedical sutures for centuries. With excellent biocompatibility, biodegradability, and sufficient supply, silk fibroin has become a popular biomaterial in tissue engineering, drug delivery, and surgery. In addition, silk fibroin can be developed into a variety of shapes with multiple structures, such as 3D porous scaffolds, films, particulates, fibers, needle patches, etc. In this study, we investigated improving the adhesive property of SF scaffold to the wet tissue with the addition of polyethylene glycol (PEG) and glycerol, and realized the sustained release of an anti-HIV protein Griffithsin.