416 Creating Novel HIV-1 gp120 and gp120Core Molecules Sharing Similar Antigenicity to the Native Trimeric Envelope Complex Using DNA Shuffling Technology X. Du*1, L. Xu1, W. Zhang1, P. Parren2, M. Zwick3, D. Burton3, R. Whalen1 1Maxygen, Inc, Redwood City, CA; 2Genmab, Utrecht, The Netherlands; and 3Scripps Res Inst, La Jolla, CA
Background: An efficient prophylactic vaccine is the ultimate solution to the rapidly emerging AIDS/HIV epidemic. Although HIV-1 gp120 is highly immunogenic, most antibodies induced by monomeric gp120 hardly recognize the trimeric envelope complex on primary isolates and fail to block viral infection. Creating novel gp120 antigens with a conformation similar to that of the trimeric Env complex may facilitate the proper exposure of the 2 potent, broadly neutralizing epitopes, b12 and 2G12 and eliminate or minimize the "decoy" effect of epitopes on wild-type gp120 monomers.
Methods: DNA shuffling technology was used to creating various shuffled libraries. Each library was then expressed in CHO-K1 cells and screened for novel antigenic phenotypes by a high throughput screening system using a dot-immunoblotting procedure and three conformation-dependent, anti-CD4BS human monoclonal antibodies: the broadly cross-neutralizing IgG1 b12 and two non-neutralizing Fab b3 and b6.
Results: Through the application of DNA shuffling technology, we have obtained a large panel of chimeric gp120 molecules classified into 4 novel antigenic categories. One interesting category showed a strong preferential reactivity to b12 and significantly weakened reactivity with non-neutralizing CD4BS mAbs, represented by b3 and b6. Immunoprecipitation studies confirmed these binding characteristics, with b12-reactivity of some clones increased up to 2.5-fold. We have also constructed wild-type gp120Core molecules through deletion of V1/V2, V3, and parts of C1 and C5. Although their reactivity to non-neutralizing mAbs b6 and b3 remains intact, their binding to b12 was significantly reduced. By applying 2 rounds of DNA shuffling, we not only recovered b12-reactivity in shuffled gp120Core molecules, but also generated novel gp120Core molecules with strong binding to b12 and significantly weakened b6 and b3 epitopes. Chimeric gp120Core molecules with other novel antigenic profiles were also identified.
Conclusions: Chimeric gp120 or gp120Core molecules with antigenicity similar to that of native envelope trimers can be generated through DNA shuffling technology. Compared to rational structure-design approaches, DNA shuffling represents a powerful technology to rapidly produce a potentially unlimited number of antigenic phenotypes of gp120. We are evaluating a number of these novel gp120 molecules in small animal models for their ability to induce broadly cross-neutralizing antibodies.