P-C5 Broadly neutralizing nanobodies selected from dromedary immune libraries with subtype C SOSIP Env glycoproteins: Optimization and preclinical development

Abstract

Nanobodies or VHH are the smallest naturally occurring antibody fragments derived from heavy chain only antibodies from Camelidae. Due to their physicochemical properties (high stability, high affinity and target specificity, extended CDR3 loops and their small size allowing to enter into protein cavities), nanobodies are very suited for preventive and therapeutic applications. We recently selected nanobodies with broad neutralizing capacity against primary HIV-1 strains of different subtypes from phage immune libraries generated from dromedaries immunized with HIV-1 subtype C gp140 SOSIP Env glycoproteins (Koch et al., 2017, in press). Two nanobodies with complementary neutralization pattern neutralized 19 out of 21 pseudoviruses in the standard TZM-bl assay. Epitope mapping data by competition ELISAs as well as negative-stain EM reconstructions with trimeric SOSIPs identified the CD4 binding site as the major target. A new selection performed on a next-generation optC SOSIP664 plus sCD4 with libraries generated at a late timepoint (7 months after the initial 7 weeks immunization cycle) identified 2 new nanobodies, which are currently being analyzed for neutralization. We further proved functionality of the nanobodies at acidic pH (as found in the vagina) and identified nanobody combinations resulting in increased breadth of neutralization in vitro. In view of preventive applications at vaginal sites of HIV-1 transmission, we are expressing the best nanobodies in a membrane-bound form and as a secreted version from lactobacilli (L. rhamnosus), which colonize the human vagina. Finally, nanobodies will be analyzed in a humanized mouse model of HIV-1 infection for their HIV-neutralizing capacity in vivo.