? CORE B The major objective for Core B (Virology/Immunology) is to support Projects 1, 2 and 3 as well as Cores C and D of this HIVRAD Program Project with extensive virology and immunology expertise. Core B will provide standardized reagents, assays and analyses in order to achieve inter-project data consistency and cross- reference. Core B will produce, characterize and standardize stocks of recombinant simian-human immunodeficiency viruses (SHIVs), SHIV-1157ipEL-p and SHIV-1157ipd3N4, for in vitro and in vivo studies of Projects 1, 2 and 3, and Cores C and D. To allow high-throughput screening, Core B will use infectious molecular clones (IMCs) containing the Renilla luciferase reporter gene (LucR IMCs) along with env genes derived from SHIV-1157ipEL and SHIV-1157ipd3N4. Core B contains a subcontract with the Institute for Research in Biomedicine (IRB), Bellinzona, Switzerland, headed by Dr. Antonio Lanzavecchia; this subcontract will generate monoclonal antibodies (mAbs) as IgG1, dimeric IgA1 (dIgA1) and dIgA2 versions for Projects 1 and 2 as well as for Cores C and D. Using standardized and uniform assays, Core B will support the Program Project by measuring humoral and cellular immune responses, including neutralizing antibody (nAb) titers in plasma and mucosal secretions. Core B will perform neutralization assays, ELISPOT assays for various cytokines, B-cell ELISPOTs, CFSE-based antigen-specific T-cell proliferation and intracellular cytokine staining. Core B also will conduct routine monitoring of all the SHIV-challenged animals for plasma viral RNA and proviral DNA loads and T-cell subsets. To provide support to all scientific components of the Program Project, Core B's Specific Aims are: 1. To produce and characterize virus stocks for in vitro assays as well as stocks of SHIV-1157ipEL-p (tier 1) and SHIV-1157ipd3N4 (tier 2) for challenge studies in rhesus monkeys (RMs). 2. To generate, produce, purify and characterize all mAbs as dIgA1, dIgA2 and IgG1 versions for passive immunization and imaging experiments in RMs. 3. To monitor humoral and cellular immune responses of experimental animals. 4. To monitor the infection status of all experimental RMs in Core C.
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