FIG 2.

Clonal expansion of recombinant vaccine strains reveals instability of antigen expression despite detectable gp120 in bulk lysates. (A) Western blot-verified expression of gp120 in three vials of lot 039 (lanes 4 to 6) and lot 040 (lanes 8 to 10) was compared to protein levels in parental BCGD transformants (lanes 3 and 7). Positive and negative gp120 controls are displayed in lanes 1 and 2, respectively. Using anti-gp120 (clone 3B3 antibody), expected bands sizes are 65 kDa and 73 kDa for lot 039 and lot 040, respectively. NS, nonspecific band. Loading controls were performed by detection of GroEL2 (clone 5177 antibody). (B) Lysates from 15 rBCGD filter clones of lot 039 were analyzed by Western blotting (lanes 4 to 18). Expression of gp120 was validated using positive and negative gp120 controls (lanes 1 and 2). Antigen expression in the clones was also compared to levels present in the parental transformant (lane 3). Expected band size is 65 kDa for lot 039. Loading controls were performed by detection of GroEL2 (clone 5177 antibody). (C) Lysates from 15 rBCGD filter clones of lot 040 were analyzed by Western blotting (lanes 3 to 17). Expression of gp120 was validated using positive and negative gp120 controls (lanes 1 and 2). The expected band size is 73 kDa for lot 040. Loading controls were performed by detection of GroEL2 (clone 5177 antibody).