Elicitation of antibody responses against the HIV-1 gp41 Membrane Proximal External Region (MPER).

摘要

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two alpha helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate alpha-helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function.;To investigate the nature of the humoral immune response in Balb/c mice directed toward the MPER, we presented the MPER segment arrayed on liposomes as immunogens to mimic its natural viral membrane context. We characterized the elicited antibody responses in sera and at the single cell level by combining a novel high-throughput microengraving screening system in conjunction with single cell PCR methods, DNA sequencing and monoclonal antibody expression rescue. Our results show that a variety of Vh and Vk genes are generated with MPER specificity among antibody-secreting bone marrow plasma cells. These antibodies demonstrate considerable somatic hypermutation and CDRH3 length variability after three immunizations. Collectively, tools are in place to investigate the B cell and plasma cell antibody repertoires elicited by such MPER/liposome vaccinations and how these can be altered by variation in immunogen design.