| With no cure on the horizon, there is widespread agreement that an HIV-1 vaccine is necessary to help slow the AIDS pandemic. A small panel of potent, broadly-neutralizing HIV-1 antibodies has been isolated from infected individuals. In passive infusion studies combinations of these antibodies can protect against viral challenge in animal models or provide therapeutic effects in humans. Our strategy towards rational vaccine design is to characterize, at an atomic level, the interactions between these antibodies and the viral proteins. Each of these antibodies has found a weakness of the virus, and understanding exactly the location and nature of this weakness would provide valuable insight into immunogen design.; The focus of my research has been the broadly neutralizing HIV-1 antibody 2G12. 2G12 was known to bind a carbohydrate-dependent epitope on the HIV-1 glycoprotein gp 120. It was unusual that one of the broadly neutralizing antibodies was recognizing a section of gp120 thought to be immunologically silent, and doing so with high affinity. In order to gain insight into the structure and function of this antibody I first determined the crystal structures of Fab 2G12 native and in complex with the disaccharide Manalpha1-2Man and the oligosaccharide Man9GlcNAc2. These structures showed that the variable heavy domains between two Fab molecules swapped positions and formed an interlocked Fab dimer. This domain-swapped structure was further supported by biochemical and biophysical evidence which showed that the domain-swapped structure of 2G12 was the functional form of the antibody. With the structure of the carbohydrates bound to the domain-swap dimer, a feasible model of the mechanism of 2G12 binding to HIV-1 gp120 was proposed. Overall, the structures have shown that 2G12 is an unexpected but elegant solution to carbohydrate cluster recognition.; After those initial discoveries, there was much interest in developing carbohydrate-based immunogens. To further dissect the carbohydrate specificity of 2G12, I determined the crystal structures of 2G12 in complex with four different oligosaccharides, ranging from Man4 to Man8. The results from these structures further elucidated the mode of binding of 2G12, showing that the antibody could bind to both the D1 and D3 arms of Man 9GlcNAc2. This knowledge of the epitope of 2G12 will help guide future design in carbohydrate-based immunogens. |
