Use of the Yellow Fever Virus for the Expression of Heterologous Epitopes
Richard Charles Garratt, Myrna C. Bonaldo, Marcos S. Freire, Mauricio Rodrigues, Ricardo Galler

One means to address some of the problems associated with new vaccine development is the use of already attenuated microorganisms for heterologous expression. In this context the yellow fever YF17D virus is an interesting candidate as it is cheap, safe, presents long-lasting immunity and has a well-defined production technology.

We have approached the question of its use in the expression of a wide range of heterologous epitopes. Potential insertion sites into the YF Envelope protein were chosen on the basis of a model for its three-dimensional structure. Initially peptides derived from the circunsporozoite protein from Plasmodium sp., corresponding to two CD8+CTL epitopes and one humoral epitope, were inserted into the fg loop of the Envelope protein. The recombinant viruses reassemble, are stable and in the case of the humoral epitope, can be neutralized by a monoclonal antibody towards it. Furthermore mouse antibodies raised to the virus recognize the parasite protein in ELISA assays.

Subsequent studies have been used to define the tolerance of the insertion site to the nature of the heterologous epitope and particularly with respect to the need to adapt to the formation of an -loop, which appears to place restrictions particularly on the pattern of hydrophobic residues.

Some of the recombinant viruses display growth restriction as well as a more attenuated phenotype for neurovirulence in monkeys. These observations are expected to be related to the fact that the insert may perturb either the dimer-to-trimer transition which occurs during infection or, more likely, the formation of rings of such trimers, necessary for the fusion of the viral membrane with that of the host. Examination of the recently determined structure for the low-pH trimer, indicates that a second insertion site, predicted in the original study may overcome these difficulties. This second locus corresponds to the glycosylation site between strands E0 and F0. Recent results on the use of this site will be presented.

Financial support: FAPESP, CNPq, FIOCRUZ