Core Facilities. Three scientific core facilities and an administrative core will provide each investigator with capabilities critical to the success of our collaborative project as described below - The Automated Cell/Biochemical Assays Core (Core A); The Protein Expression Core (Core B); The Animal Services Core (Core C); The Administrative Core (Core D).
Core A - The Automated Cell/Biochemical Assays Core
Core A, Automated Cell/Biochemical Assays Core designs and carries out cell-based and biochemical assays to characterize the specificity, efficacy, and potency of HIV-neutralizing Abs. We have adapted and validated the standard in vitro pseudovirus neutralization assay for execution by a liquid handing robot. Core A personnel validate, refine, and troubleshoot protocols for automating the manual in vitro neutralization assays and then perform automated in vitro neutralization assays for evaluating Abs for Drs. Nussenzweig, Ravetch, and Bjorkman. In addition, Core A performs rapid and high-throughput binding assays to assess the affinities of Abs for Env proteins, as required for Drs. Nussenzweig and Bjorkman, and affinities of modified Fc regions for Fc receptors, as required by Dr. Ravetch. Core A is led by Dr. Jost Vielmetter, who has many years of experience in assay automation through his background in biotech and his work with the automated assay station at Caltech’s Protein Expression Center.
Core B - The Protein Expression Core
Core B, Protein Expression Core provides access to purified proteins for biochemical, structural, and efficacy assessments. Expression and production of purified proteins including HIV proteins, Fc receptors, and natural and designed Abs, is a major component of our program project. Dr. Nussenzweig requires mutant HIV proteins for epitope mapping. Dr. Ravetch needs a panel of CD4bs and other anti-HIV Abs with modified Fc regions and altered glycans for evaluating effector functions in Project 2. Dr. Bjorkman needs large quantities of purified Fabs, modified Fc regions, Fc receptors, and many forms of gp120 antigens for complex formation, crystallization, structure determinations, and neutralization assays in Project 3. The large number of different Abs, HIV proteins, and Fc receptors to be expressed and purified is greatly facilitated by a dedicated protein expression core. Core B is led by Dr. Bjorkman, who has served as the faculty advisor for the Caltech Protein Expression Center for >15 years and works closely with Dr. Vielmetter to coordinate protein expression, purification, and assay automation.
Core C - Animal Services Core
Core C, Animal Services Core produces, maintains and breeds mice for in vivo experiments. Two novel mouse strains are being used or developed for this program, the in vivo TZM-bl mouse, described by Nussenzweig, and the fully FcR humanized mouse, described by Ravetch. These strains will be crossed to provide a strain that will provide a means of assessing the in vivo effector components impacting on fully human bNAbs by interrogating both gp160 epitopes and Fc effector activity for the novel and re-engineered bNAbs. In addition, the core will utilize human IgH knock-in mice and produce humanized mice for HIV infection experiments. The latter will require breeding of 30 breeding pairs of NOD/RAG/g mice, and maintaining the progeny for 10 weeks while they develop human immune systems and are infected with HIV-1. Large numbers of mice are required because reconstitution is variable from 5-25% and infection is also variable depending on the reconstitution. Generating and maintaining this unique resource requires a dedicated animal core involved in breeding, screening, and testing these animals. Drs. Nussenzweig and Ravetch rely on this core extensively to evaluate neutralization and effector functions of bNAbs, and Dr. Bjorkman uses this core to evaluate designed bNAbs and designed immunogens. Core C is led by Dr. Nussenzweig who has >25 years of experience working with genetically engineered mouse models and has served for 10 years as the chair of the Rockefeller University Animal Care and Use Committee.
Core D - The Administrative Core
Core D, Administrative Core will carry out functions that interface with the projects in the Nussenzweig, Ravetch, and Bjorkman laboratories and with the three scientific cores, providing efficient organization and communication. The Administrative Core arranges meetings among investigators, including monthly video-conferences to present results; prepares protocol submissions and annual renewals to Institutional Review Boards; coordinates material transfer agreements; prepares reports and helps to prepare, submit and track manuscripts; assists in shipping reagents between Caltech and Rockefeller; monitors budgets; and manages personnel including immigration, stipends, benefits and housing. Core D is led by Dr. Bjorkman with help from her administrative assistant, Ms. Marta Murphy.