Annual and Interim Progress Report Summaries
Principal Investigator: Shui-Lok Hu
Project: Unmasking Conserved Epitopes
Submitted August 1, 2012
We are generating Env with the N197 glycan mutant (N7) from diverse HIV-1, including multiple subtype B isolates, early isolates of subtype C (1157i and 1084i), and a subtype A isolate (QA255) from a Kenyan woman who developed broadly-neutralizing antibodies. Increased neutralization sensitivity to CD4-binding site antibodies is observed for all N7 mutants tested. Studies are underway to compare the breadth and potency of neutralizing antibody response in rabbits primed with recombinant vaccinia virus expressing 89.6 WT or N7 Env and boosted with gp160 or gp120. Results will inform the design of studies evaluating the effect of prime-boost immunization with heterologous Env.
We are also generating recombinant vaccinia viruses expressing glycan mutants in V2 proximal to the integrin binding site of the Env from an early isolate 1084i. These will be used to evaluate the role of these glycans in modulating Env-integrin interactions and immunogenicity.
To assess the contribution of CD4-independence to Env immunogenicity, we derived variants of SHIV 89.6 (WT and N7 versions) and HIV-1 R3A and QA255 that could replicate robustly in CD4-/CCR5+ T-cells. Env clones have been generated that mediate CD4-independent fusion efficiently. We are assessing the genetic basis for this phenotype, their effect on neutralization sensitivity, and their functions in replication-competent viruses. These novel Envs provide a platform to determine the extent to which CD4-independence may alter immunogenicity and breadth of neutralization responses.
We have completed the evaluation of Env cytoplasmic tail modifications in R3A, 89.6, QA255 and JR-FL, and have identified mutations that increase the surface expression approximately 10-fold over the wild-type. Immunogenicity of a CD4-independent R3A Env with these tail modifications is being determined in huCD4 mice. Our observation that DCs limit Env surface expression and that this effect can be overcome by our tail modifications is unexpected and potentially important for vaccine design.