Cell-surface Display and Panning of HIV-1 Derived Envelope Proteins

P41.27

Background: Available display systems allow the screening of millions of candidate proteins and are the method of choice to identify optimized antigen-antibody binding.

We established a mammalian cell-surface display to present HIV-1 envelope derivatives in a natural, trimeric and membrane bound environment. This allows us to generate affinity enhanced envelope derivatives against broadly neutralizing antibodies (bNAbs) in order to select potent Envelope (Env) based vaccine candidates.

Methods: An HIV-1 derived lentiviral vector was developed to infect HEK293T cells at a low multiplicity of infection (MOI), in order to correlate phenotype and genotype. The vector was designed and proven to express both GFP and Env in a constant relationship, enabling indirect normalization for Env expression by detecting GFP. After staining with an appropriate bNAb, Env-displaying cells were selected for high affinity binding via FACS-Sorting.

To adapt this system to the use of very large libraries, a refined vector system was developed, allowing the stable genomic integration of both ENV and GFP at a distinct integration site. This ensures that only one envelope variant is expressed per cell, efficiently linking phenotype and genotype. Due to the stringent linkage of ENV and GFP, GFP expression can again be used as a means to normalize for Env expression in the FACS-Sorting process.

Results: A small model library of Env variants with distinct binding capacities towards the bNAb 447-52D was used to evaluate both techniques.

The proof-of-concept experiment for the virus-based approach led to an enrichment of up to tenfold for the Env variant with the highest affinity toward 447-52D after two rounds of selection.

Using the stable cell line technology, the desired Env high affinity variant could even be enriched up to 39-fold after only one round of FACS-Sorting.

Conclusions: Hence, these techniques provide the possibility to screen for membrane bound Env variants with high binding capacities towards any bNAb applied.