Due to the complex nature of the steroidogenic pathway, which involves enzymes with multiple substrates as well as multiple enzymes acting upon the same substrates, rigorous methods for molecularly dissecting this pathway are required for understanding it’s role in AR signaling and prostate cancer. Over the course of current androgen deprivation treatments, many patients develop castration resistant prostate cancer (CRPC). When the disease reaches this stage it has been known that cancer utilizes multiple pathways, known as the backdoor or bypass pathways, in order to continue synthesizing the androgens which ultimately drive cancer progression. To learn more about these alternate pathways, isoform-specific CRISPR/Cas9-mediated knockouts of these highly similar genes can be used to determine the specific contributions of each of these factors to de novo androgen synthesis and androgen scavenging in prostate cancer tumors. These CRISPR constructs will be used to generate gene specific knockout prostate cancer cell lines. Validation of each line will be done using a T7 endonuclease assay followed by western blot to confirm knockout of the protein. Further analyses using these specific knockout cell lines will provide insight on which genes are involved in the new pathway when the classical pathway is shunted.

A more complete understanding of a protective HIV-specific immune response is critical to designing effective vaccine strategies. Researchers have characterized hundreds of antibodies (Abs) that elicit broad and potent neutralizing activity but much less is known about Abs that mediate FcR-dependent responses, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular virus inhibition (ADCVI) activity. ADCC involves antibody-dependent recruitment of natural killer cells through Fc/FcR-dependent interactions and subsequent lysis of infected cells whereas ADCVI activity includes ADCC but also involves chemokine release and FcR-mediated phagocytosis. Intriguing evidence from the recent RV144 vaccine trial demonstrated a modest 31.2% protection that was subsequently correlated with higher levels of ADCC activity, rendering previously uncharacterized ADCC- and ADCVI-mediating Abs of increased interest. Here we describe 13 HIV-specific Abs identified from an individual who developed a uniquely broad and potent HIV-specific response early following infection. All 13 Abs were tested for neutralizing and ADCC activity and a small subset were tested for ADCVI activity. While only two Abs demonstrated modest neutralizing activity, two Abs demonstrated broad, cross-clade ADCC activity as well as ADCVI activity. Our project aims to firstly evaluate all 13 HIV-specific Abs for ADCVI activity and secondly, to map the binding properties of the mAbs identified to mediate ADCVI. These latter studies will be completed first using a high-throughput binding antibody multiplex assay (BAMA), and secondly through a more refined series of competition-based enzyme-linked immunosorbent assays (ELISAs). Preliminary analysis has indicated that at least one of these 13 Abs mediates ADCVI but neither neutralizing nor ADCC activity and mapping studies suggest this Ab may bind to the gp41 portion of the HIV envelope. Further understanding of the epitope specificities of Abs that mediate ADCVI activity will help us characterize how this individual mounted a broad and potent response early following infection.