Structural Approaches To HIV-1 Immunogen Design For BNAb Generation

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Broadly neutralizing antibodies (BNAb) to HIV-1 primarily target the membrane proximal ectodomain region (MPER) of the viral gp160 envelope protein. We have studied the MPER segment in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and surface plasmon resonance (SPR) methodologies. Structural analyses reveal a tilted N-terminal a-helix (aa 664-672) connected via a short hinge (673-674) to a flat C-terminal helical segment (675-683) that collectively forms a metastable L-shaped structure immersed in the membrane. The 4E10 BNAb extracts buried W672 and F673 following initial encounter with the surface-embedded MPER. Given conservation of this tryptophan-rich sequence in HIV-1, HIV-2 and SIV, the data have implications for vaccine design and suggest how BNAbs perturb tryptophan residue-associated viral fusion involving the MPER. Here we shall examine how other BNAbs such as Z13e1, 2F5 or newly generated MPER binding antibodies induce conformational change around W672 and F673 or elsewhere in the segment, and whether such structural changes upon antibody binding are linked to viral neutralization. Moreover, specificity and diversity of antibodies arising during natural HIV-1 infection versus elicited upon vaccination will be compared. Sensitive EPR residue depth and inter- residue distance measurements will allow for relatively rapid screening of detectable changes in MPER conformation. Potency of neutralization will be correlated with MPER structural changes. Once identified by EPR, interaction will be followed by detailed NMR analysis. How lipid constituents of the virosome, including cholesterol, affect the membrane-embedded structure of the MPER, or its ability to undergo conformational changes upon antibody binding, will be assessed. In addition, lipid-enveloped nanoparticles as carriers of natively configured MPER segments with a bioresorbable poly (lactide-co-glycolide) (PLGA) core harboring invariant and promiscuous class II MHC molecule-binding epitopes for stimulating CD4+ T cell activation will be tested in murine intradermal immunization studies aimed at eliciting BNAbs. These nanoparticles will be further armed with immune activating adducts to optimize the magnitude of the specific immune response. ELISA, BIAcore and antibody neutralization assays will assess the breadth of neutralizing responses and determine the optimal number of distinct MPER sequences needed to induce antibodies capable of neutralizing clade B isolates. Given that globally, to date, 65 million human infections with HIV-1 have been estimated, the development of a vaccine eliciting broadly neutralizing antibodies in normal subjects will be an enormous preventive advance in the fight against AIDS.