Binding of Antibodies and their Fragments to a Suitable DENV Antigen

Our project concentrated on finding an appropriate therapeutic for Dengue, a disease that severely affects Southeast Asia.

While it would have been impossible to develop a complete drug with our time and available resources, we tried to accomplish as much as we could by proving that our protein would bind to the dengue antigen, and that it would be able to neutralise it effectively. Since we were developing a novel antibody, there were two parts to Proof of Concept - the first was to ensure that our antibody was getting expressed and was properly folded, the second was to determine that it was binding to and neutralising our antigen.

After we cloned our gene into the vector and verified it using a confirmatory restriction digest, we transformed the same into SHuffle E.coli cells, cultured the bacteria, and induced protein expression with IPTG.
We spun the cells down following this, and lysed them using sonication. We spun down the lysate and used the supernatant for downstream purposes.

We optimised expression of the scFv using statistical Design of Experiments, running 15 conditions to check the best one for soluble expression of our protein. Analysis was performed using SDS-PAGE gels, standardised using BSA standards, and controlled against the expression of the empty vector backbone.

Proving Expression

We expressed both the scFv and the scFv with our linker peptide in SHuffle E.coli, verifying expression with a Western Blot using anti-His antibodies. Anti-rabbit HRP conjugates were used as secondary antibodies and chemiluminescence was used to image the substrate-linked antibody.

We successfully pulled down the scFv with the FcRn binding peptide (scFv+FcRnBp) with Ni-NTA resin using elutions with 500mM imidazole.
We further concentrated this protein using a centricon to a final concentration of 12.4µM.

Proving Proper Folding

We ran a Thermal Shift Assay with SYPRO Orange dye to verify that our protein was folded correctly.

SYPRO Orange is a dye that binds only to the hydrophobic residues of a protein, so it binds only when the hydrophobic residues are exposed i.e. when the protein is unfolded.

We used the dye to determine the unfolding of the protein over a range of temperatures, and plot a melt curve for the protein.

The melting temperature of the NeoFv was around 31 C in the buffer we used.
This assay indicates that the protein is folding properly in solution. It is important to run the assay in different buffers, however, to compare the melting temperature in each of them.

Proving the Binding and Neutralization

For the second part of the Proof-of-Concept, we had to demonstrate that NeoFv was functional and capable of neutralising the dengue virus. To do this, we ran a VLP Fusion assay.

Vacuoles are filled with dye to concentrations so high that the colour is extinguished.Virus-like Particles are added to the vacuoles and the pH is lowered to make the VLPs fuse with the vacuoles. This leads to the dilution of the dye, and increase in visible colour. VLPs were incubated with NeoFv and were added to the vacuoles, with the expectation that a neutralising interaction between the two would decrease the colorimetric readout, due to NeoFv inhibiting membrane fusion.

Our protein showed significant neutralisation of the virus, at high concentrations.
The 90% control marker represents inhibition of fusion with no antibody added. The NeoFv, at 5X dilution from the stock, shows a 57% drop from the control line - to 33% fusion.
This inhibition drops to 5% at 50000X dilution.

We cannot accurately reason why the protein was not able to neutralise the virus completely but the protein might not have been at optimum functionality due to inadequate shipment conditions. Additionally, the assay wasn’t optimised for our protein due to a lack of time and resources, which might have contributed to our results.