To study the feasibility of developing HAV as an expression vector, synthetic oligonucleotides coding for a polylinker a HAV protease 3Cpro cleavage site was inserted into different parts of the coding sequence of the HAV cDNA. A construct which contained the polylinker at the N-terminus of the HAV polyprotein was infectious and named HAVvec1. Insertion of the same polylinker into other parts of the HAV genome killed the virus. Synthetic oligonucleotides coding for the octapeptide FLAG was inserted into the polylinker of HAVvec1. Viable HAV expressing the FLAG epitope was produced upon transfection of HAV cDNA into FRhK/4 cells. Sedimentation through sucrose gradients and Western blot analysis showed that the FLAG peptide was predominantly expressed in empty capsids fused to VP0 and forming a PreVP0 protein. Several other antigenic peptides such as the main epitope of the hepatitis B virus surface antigen and the NANP epitope of malaria have been expressed using HAVvec1. We are currently cloning larger reporter genes such as CAT and GFP into the infectious cDNA of HAV to determine whether larger molecules can be expressed using this system. Two killed vaccines against HAV were recently approved by FDA. . PLA supplements and combination vaccines containing inactivated HAV vaccine are currently under review. These killed vaccines appear to be highly effective but suffer from being very expensive to produce due in part to the poor replication of HAV in cell culture. This research project concerns ways to improve the replication of HAV in cell culture. It also relates to the development of live, attenuated vaccines that would require a much lower viral content and would therefore be cheaper. Live vaccines may also be more immunogenic and additional ways to improve immunogenicity are being explored. Molecular cloning of reporter genes into the HAV genome will lead to new ways of evaluating potency of the present kill vaccines and future attenuated vaccines. This research could also lead to the development of combination vaccines in which the expression of different epitopes such as the HBsAg are directed by the HAV genome.
