Protein Core will be the provider of all the important reagents for Project 1 and Project 2 of this grant proposal. It will use the information coming from Project 4 to generate HIV Envelope trimers that will be used as baits for identification of novel neutralizing antibodies in Project 1 and 2. The number of baits generated by Protein Core will be 10-20 per patient. The amount will vary depending on the need of the researchers in other projects. The Core has the capacity to produce up to 100 mg of trimers. With the progress of the Program Protein Core will generate neutralizing antibodies identified with the help of these baits. There will be two dozen antibodies generated per patient. Protein Core has experstise to do cell culture necessary to generate all these proteins. The Core has successfully purifed several grams of a broadly neutralizing antibody that was used in a SHIV protection experiment in non-human primates. Protein Core will perform transient transfections and use mammalian cells, HEK 293T and CHO, to produce the proteins, then we will perform affinity chromatography to purify the proteins for further use by other researchers or further characterization. Protein Core will do some initial characterization of the neutralizing antibodies by BIAcore, the technology that allows real time sudy of protein interaction. We will study the affinity of novel antibodies toward various HIV Envelope proteins.
Specific Aims Aim 1. Preparation of Envelope constructs in particular gpl40 trimers that will act as baits for identification of neutralizing antibodies and tools for mapping serum neutralizing activity and antibody characterization.
Aim 2. Expression and purification of novel antibodies in particular neutralizing antibodies identified in Projects land 2.
Aim 3. Biochemical characterization of the novel antibodies.
Neutralizing antibodies are important for a successful HIV vaccine. Protein Core will produce reagents based on the virus Envelope proteins that will serve as baits for identification of these antibodies. After that the Portein Core will produce neutralizing antibodies for further study and characterization by other researchers.
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