Introduction: Recently introduced antiviral therapies have proven clinically effective against HCV, however issues such as the high costs associated with these drugs, frequent undiagnosed infection and re-infection after treatment suggest an effective vaccine will be required to quell the epidemic. A major barrier for HCV vaccine development is the high sequence variability of the E2 glycoprotein, which is the major target of neutralizing antibodies. We have developed the Delta3 immunogen by removing three regions with high variability. Delta3 induces antibodies able to neutralize a broader range of HCV strains, and this is particularly true of a disulfide-linked high molecular weight (HMW) form of Delta3. We are defining features of the disulfide-bonding arrangement and glycan composition which distinguish Delta3 from other E2 species and Delta3 HMW from other Delta3 forms that can be used as quality control parameters for future vaccine manufacture.
Methods: Delta3 was expressed by either transient or stable transfection of 293F and CHO DG44 cells, and purified using metal affinity and gel filtration chromatography. Disulfide bonds of Delta3 were determined by nanoLC ESI MS/MS using deglycosylated and protease digested protein. Glycan composition was examined using lectin array.
Results and Discussion: Good yields of Delta3 were obtained in stable 293F cells. For the H77c strain, 5 mg/100 ml of tissue culture supernatant was achieved for the monomeric form. The HMW yield was approximately10-fold lower. Other HCV strains produced higher HMW:Monomer Delta3 ratios and these HMW forms showed similar patterns of antigenicity as H77c HMW, however overall yield was lower. Delta3 disulfide linkages not observed in the crystal-derived structures of other E2 forms have been identified. Additionally, several lectins have higher affinity for different Delta3 quaternary forms, indicating a potential role for glycan composition in oligomer formation or vice versa. We are now in a strong position to use these tools to validate Delta3 production prior to use in clinical trials.