The purpose of the Molecular Biology Core is to assist the HIVRAD program in characterizing the epitopes recognized by MAbs and polyclonal human and monkey antisera that target QNEs. The main role of this Core will be to generate DNA and protein reagents needed by the other projects and the Viral Immunology Core.
The Specific Aims are : 1) To design and produce plasmids expressing chimeric and mutant HIV-1 Envs for epitope mapping studies and for viral neutralization assays in Projects 1, 2 and 4. 2) To generate monomeric WT gp120 and gp140 proteins, and soluble monomers and trimers that express QNEs. These proteins will be used for binding and adsorption studies in Projects 1, 2 and 4, and as immunogens in Project 3. These studies willl be guided by results of epitope mapping experiments obtained from Projects 1, 2 and 4. 3) To generate infectious SHIV pseudotypes and infectious mutant SHIVs with variant Envs that express QNEs. Fragments of the SHIV Env will be cloned into appropriate vectors to facilitate mutagenesis experiments, and the complete SHIV Env-Rev genes will be subcloned into the pcDNA 3.1 vector to generate viral pseudotype. These will be used to allow efficient construction of Env mutants and chimeras and for the generation of viral pseudotypes for the characterization of functional activity of the mutant SHIV Envs. QNEs defined in Projects 1 and 2 will be introduced into the SHIV1157ip Env for characterization of their infectivity in monkey blood PBMCs and their neutralization sensitivity. Ultimately, mutant SHIV Envs will be introduced in the SHIV1157ip proviral backbone to generate infectious SHIVs expressing QNEs for invivo studies described in Project 3. 4) To isolate and characterize escape mutant Envs from infected macaques. The evolution of Env sequence in the infected animals will be investigated during the course of infection to follow the anti-QNEs neutralizing responses and identify viral escape mutants. Envs will be cloned and escape mutants will be identified by neutralization assays (Viral Immunology core).
The goal of this HIVRAD program is to define and characterize new bnAbs that target QNEs. The molecular biology core will play a primordial role in providing molecular reagents to the other projects and the viral immunology core. The availability of a centralized facility will increase the efficiency and improve the uniformity of reagents and subsequent data.
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