Improving the immunogenicity of recombinant adeno-associated virus

Abstract

Recombinant adeno-associated virus (rAAV) has many attractive features as a T cell vaccine vector, however disappointing immunogenicity was seen in a phase I clinical trial. Subsequent in vivo studies showed that several pseudotypes of rAAV elicited a dysfunctional CD8+ T cell response in mice that may reflect poor priming, lack of CD4+ T cell help or persistence of the vector. Dendritic cells are the key antigen presenting cells that prime a naive T cell response. Therefore, in this study multiple pseudotypes of rAAV were screened for their ability to transduce human monocytederived dendritic cells (MoDCs) and rAAV2/6 identified as the optimal pseudotype. Further improvements in transduction efficiency were achieved by mutation of surface-exposed tyrosine 731; analogous mutations in the capsid of AAV2 have previously been shown to decrease proteasomal degradation and increase nuclear trafficking of rAAV. Lysine 531 was identified as a key residue in the tropism of pseudotype 6 for MoDCs. Interestingly, while this residue is critical for the interaction with immobilised heparin, soluble heparin did not inhibit the transduction of MoDCs by AAV6. rAAV is a poor maturation stimulus of MoDCs therefore the development of immunoreactive vectors was a major goal of this project. One strategy adopted was the generation of vectors that encode MAVS, a signalling molecule in the RIG-I/MDA-5 pathway whose expression led to robust activation of NFκB and IRF-3 signalling pathways. However yields of vector encoding MAVS were greatly reduced due to reduced viability of the producer cell line. Therefore alternative means of vector production were sought. As rAAV has previously been successfully produced in insect cells and insects lack a homologue of MAVS, a baculovirus production system was developed for the production of pseudotype 6 rAAV. Successful production of rAAV was demonstrated, however further optimisation is required to allow production of high titres of rAAV necessary to facilitate further assessment of self-adjuvanting pseudotype 6 rAAV as a vaccine vector.