540. Retroviral Mediated Gene Transfer of Hexb in Sandhoff Disease

Abstract

Top of pageAbstract Vaccines that stimulate broadly neutralizing antibodies to HIV-1 will have a major impact on reducing transmission. However this remains an elusive goal because the antibodies induced by the envelope glycoprotein (Env) do not effectively neutralize primary isolates or viral strains that are important in transmission. Using DNA shuffling and screening technologies, we had identified novel gp120 variants with preferential reactivity to the neutralizing human mAb b12 and significantly weakened binding to non-neutralizing CD4BS-binding mAbs b3 and b6. This phenotype mimics that of the native trimeric envelope complex. Biacore studies of mAb-gp120 interaction demonstrated that the enhanced affinity of b12 in the shuffled variants resulted largely from a decrease in the dissociation rate. DNA shuffling of the 'core' form of gp120, which lacks V1/V2, V3 and parts of C1 and C5, also generated gp120 variants that possessed these antigenic properties. We immunized rabbits with >100 independent gp120 variants using electroporation-mediated DNA vaccination, followed by boosting with a heterologous or homologous recombinant protein antigen. A single-cycle pseudovirus-based assay was used to evaluate the HIV-1 neutralization activities of purified immunoglobulins. About 60% of the full-length gp120 variants induced neutralizing antibodies to the TCLA strain SF162, indicating that the DNA shuffling and screening approach provides novel variants that have gp120-like immunogenicity. Several of the shuffled variants induced antibodies to primary viral isolates and five clones were able to neutralize eight primary isolates in addition to TCLA strains. These shuffled variants are candidates for additional shuffling to screen for further improvements in immunogenicity and for testing using additional vaccine formats, including optimized immunization protocols. DNA shuffling extensively recombines mixtures of parental genes such as the Env coding sequences to create highly functional libraries of shuffled chimeras. We have successfully applied this technology to evolve novel viral phenotypes, to improve the antigenicity of the Hepatitis B virus envelope protein and to generate tetravalent Dengue virus antigens. The application of DNA shuffling to HIV vaccine development provides a new approach for generating novel Env structures as vaccine candidates.