Core A Summary
The ability to elicit potent and broadly neutralizing antibodies (bNAbs) against HIV-1 through vaccination is one of the prime challenges of biomedical research in our time. The proposed HIVRAD project aims to address this challenge. Our approach is based on the concept that immunogens that bind to inferred germline Abs (iGLs) that are precursors to known mature bNAbs can initiate an Ab response that can be guided to produce bNAbs using a series of sequential immunogens. Therefore the focus of this grant is to evaluate immunization regimens in animal models (Project 1) using immunogens selected by structure-based design and library screening (Project 2). Key requirements for the success of this project include the ability to evaluate thousands of serum samples from immunized animals to determine whether they are producing specific and productive responses, producing and screening thousands of potential immunogens and Abs, and assessing binding and neutralization potency and breadth. Here we describe a scientific core dedicated to using liquid handling robots for automated expression of small quantities of many different proteins and high-throughput automated assays. The Cell/Biochemical Assay Automation Core will design and carry out automated cell-based and biochemical assays to express proteins in high-throughput, characterize the binding affinity and specificity, and evaluate the potency of HIV-neutralizing antibodies. Core A personnel will validate, refine, and trouble-shoot protocols for the automated protein expression, binding, and in vitro neutralization assays and perform assays for evaluating antibodies and Env proteins for Drs. Nussenzweig and Bjorkman. The core will train and assist students, postdoctoral fellows, research assistants, and investigators in the analysis and interpretation of data from automated assays. The Automated Cell/Biochemical Assays Core will maintain two custom-equipped Evo Freedom Liquid handling stations and a surface plasmon resonance instrument with high throughput capabilities for the automated assays proposed in this project.
Core B summary
Biochemical, structural, and immunization experiments to evaluate new immunogens designed and selected to induce broadly neutralizing antibodies will require expression and purification of large numbers of proteins, including HIV envelope proteins and antibodies. The Protein Expression Core will provide purified proteins for this program project, which will facilitate the goals of identifying immunogens and defining immunization strategies that elicit a strong and targeted immune response. Antibodies isolated from immunized mice (Project 1) will be expressed and purified for binding and biochemical characterization as well as for structural studies (Project 2). Env proteins, both monomeric gp120s and native-like Env gp140 trimers (SOSIP.664 constructs), will be expressed for immunizations in Project 1, binding studies in Projects 1 and 2, and structural/biochemical analyses in Project 2. Core B will use various expression systems to produce Env proteins with different glycosylation to determine the effects of glycosylation form on the generated immune response. The large number of different antibodies and HIV proteins to be expressed and purified for this program requires a dedicated protein expression core.
Core C Summary
The Administrative Core (Core C) will provide organizational and administrative support to facilitate communications, reporting, budget management, and other tasks that interface with the two projects and two scientific cores. The administrative core will include Dr. Pamela J. Bjorkman as Core Leader, her Administrative Assistant, a Research Scientist in her lab, the Lab Administrator in Dr. Michel Nussenzweig’s lab, and the Administrative Assistant for Dr. Jost Vielmetter, the Core A and Core B Leader. The Administrative Core will 1) Organize and govern the HIVRAD project and 2) Coordinate the interactions within the HIVRAD project and with the broader scientific community. This includes arranging formal communications among investigators, preparing protocol submissions and annual renewals to the Institutional Review Boards, coordinating material transfer agreements, preparing reports and manuscripts, monitoring budgets, planning annual meetings and monthly conference calls, maintaining the HIVRAD website, and generally overseeing the scientific and administrative needs of the Program Project. In addition, the Administrative Core staff will assist in the effective sharing of data by maintaining shared cloud-based notebooks and databases.