Figure 1
Design of rubella vectors, their replication and expression of inserts at the structural site. (A) The non-structural genes are located near the 5′-end of the genome (blue) and are controlled by the genomic promoter (Pgen). The structural genes are located near the 3′ end (yellow) and are expressed as a structural polyprotein controlled by the strong subgenomic promoter (Psub). The structural insertion site is located between envelope glycoproteins E2 and E1. The antigenic insert (Ag, orange) is flanked on both ends by a transmembrane domain (red or green) and signal peptidase cleavage sequence E1SP (blue). The structural polyprotein is cleaved at three sites by signal peptidase (red arrows) to release mature structural proteins and the insert. (B) Replication of rubella vectors was demonstrated by Western blot of infected Vero cells with antibodies to rubella proteins E1 and C (left panel). Expression of the MPER-HIVTM insert was detected with monoclonal 2F5 (middle panel), while expression of the BC-sGag2-E2TM insert was detected with polyclonal HIV immune globulin (right panel). The control vaccine strain RA27/3 showed good growth (left panel), but no expression of either antigen (middle and right panels). The rubella-MPER vector replicated well (left panel), while expressing MPER-HIVTM as a 10 kDa band (middle panel, yellow arrowhead). The rubella-BC-sGag2 vector also replicated well (left panel) and expressed Gag as a 14 kDa band right panel, green arrowhead). Control lanes include AT-2 inactivated SHIV virions for gp41 (lane SHIV, red arrowhead), recombinant p55 SIV Gag protein (lane p55, blue arrowhead), and uninfected cells (lanes N).