Cohesin Fusion Proteins for HIV Env Antibody Evaluation

Cohesin fusion proteins [30 (link)] were expressed in CHO-S cells from vectors containing the above Env gp140 sequence, or Env fragments (gp120 residues 94–1550; gp41 residues 1551–2064, inserted between the cohesin domain and 6 C-terminal His codons,). HIV Env-specific IgG antibody titers in serum from vaccinated animals were assessed at indicated time points post-immunization using ELISA. ELISA plates were coated with 2 μg/ml of the cohesin fusion proteins in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4. Serial dilutions of serum starting at 1:500 in TBS blocking solution (StartingBlock T20, Pierce) were incubated in the wells overnight at 4°C. After washing, plates were incubated with HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) in TBS blocking solution (Thermo Scientific, Rockford, IL) for 2 h at 37°C, then washed and developed with HRP substrate (TMB, Life Technologies), stopped with equal volume of 1N HCl and read at 450 nm. The log₁₀ transformed and normalized areas under the curves (AUC) data were calculated for each animal at each time point using GraphPad Prism 6 software (GraphPad, La Jolla, CA). EC50 calculations were based on Log10 transformed and normalized data with non-linear regression curve fit using sigmoidal dose response with variable slope constraints.