Development of mammalian cell-display and panning techniques for the selection of HIV-1 vaccine candidates

An ever-increasing number of broadly neutralizing antibodies (bNAb) against the HIV-1 envelope (Env) have been discovered so far. Unfortunately, most antibody responses against HIV-1 in an acute infection are only strain-specific and quickly bypassed by escape mutations. Vaccination trials designed to re-elicit bNAbs that mediate protective immunity have remained futile (reverse vaccinology). The neutralization activity of the discovered bNAbs has been attributed to their affinity to functional Env trimers, although each cross-reacts with non- trimeric, immature or otherwise non-functional Env molecules. Therefore, a discovery platform was developed in order to select bNAb-bound, native, trimeric envelope variants that are displayed on the surface of mammalian cells. Conclusively, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells by a low multiplicity of infection (MOI), in order to link phenotype and genotype. Hence, infected cells expressed and displayed membrane-associated Env proteins on their surface (lenti-cellular display). Subsequently to staining with an appropriate monoclonal antibody, Env-displaying cells were selected for high affinity binding via FACS sorting (Panning). A small model-library of Env variants with distinct binding capacities against the bNAb 447-52D was generated for evaluation. In a proof-of-concept study, it was demonstrated that the Env variant with the highest affinity to the applied bNAb was enriched in two rounds of selection (up to 10-fold). Hence, the demonstrated technique provides the possibility to screen membrane-bound Env variants for high binding capacities against the bNAb applied. Nevertheless, the presented lenti-cellular display and FACS-mediated panning platform still needs further optimizations. Prospectively, it needs to be challenged by libraries of larger diversity and selected candidates should be evaluated in vaccination trials.