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.