We have investigated the use of soluble chimeric trimers of the major
capsid protein VP7 of African horse sickness virus (AHSV) as a
vaccine delivery system by targeting some of the natural hydrophilic loops on the VP7 top domain for the insertion of foreign
peptides. Key to this trimer display strategy is the solubility of AHSV VP7 and how the solubility of this hydrophobic
protein can be manipulated by inserting
peptides into the top domain. To investigate, we generated different cloning vectors by inserting multiple cloning sites at three different positions in the VP7 gene. These modifications inserted six
amino acids at the cloning sites and in some cases this converted VP7 to a largely soluble
protein without affecting the ability of the modified
proteins to form trimers. The vectors were used to generate a number of soluble VP7 fusion
proteins including a fusion with a 36
amino acid insert that overlaps important immunological domains on
protein VP1 of foot and mouth disease virus (FMDV) as well as a 110
amino acid peptide derived from AHSV VP2. Soluble trimers of these fusion
proteins were able to elicit a good insert-specific immune response in guinea pigs.
l-Arginine was found to reverse
protein aggregation and was employed as an effective strategy to isolate relatively pure soluble chimeric VP7 trimers. Another factor that increased VP7 solubility in both wild-type VP7 and one of the VP7 vector
proteins was the substitution of the
leucine residue in position 345 of the VP7 C-terminus with a hydrophilic
arginine residue.