92BR
60 40 20
10-4
10-2
100
H330A N229R T244S V85A N332A WT
80 60 40 20 0 10-6
102
10-4
H330A K46R N229R R419A T244S V85V BR N332A WT
60 40 20
10-4
10-2
100
Per Cent Neutralization
Per Cent Neutralization
92BR
80
0 10-6
102
H330A K46R N229R R419A T244S V85V N332A WT
60 40 20
10-2
100
PGT-123 (ng/μL)
102
Per Cent Neutralization
Per Cent Neutralization
92BR
10-4
60 40 20 0 10-6
10-4
H330A N229R T244S V85A N332A WT
80 60 40 20 0 10-6
10-4
10-2
100
H330A N229R T244S V85A N332A WT
60 40 20
10-4
10-2
100
PGT-123 (ng/μL)
102
H330A K46R N229R R419A T244S V85A N332A WT
80 60 40 20 0 10-6
10-4
10-2
100
102
PGT-122 (ng/μL)
80
0 10-6
100
JRCSF
100
102
IAVIC22
100
10-2
PGT-121 (ng/μL)
PGT-122 (ng/μL)
80
0 10-6
H330A K46R N229R R419A T244S V85A N332A WT
80
102
IAVIC22
100
PGT-122 (ng/μL)
100
100
JRCSF
100
PGT-121 (ng/μL)
PGT-121 (ng/μL)
100
10-2
Per Cent Neutralization
0 10-6
IAVIC22
100
Per Cent Neutralization
H330A K46R N229R R419A T244S V85V N332A WT
80
102
Per Cent Neutralization
100
Per Cent Neutralization
Per Cent Neutralization
4.3 results
JRCSF
100
H330A K46R N229R R419A T244S V85A N332A WT
80 60 40 20 0 10-6
10-4
10-2
100
102
PGT-123 (ng/μL)
Figure 4.6: Single Mutant Neutralization Curves Single point mutations were introduced into plasmids containing the HIV Env gene using site-directed mutagenesis. Mutant Env plasmids were then cotransfected with plasmids containing an Env-deleted HIV genome backbone (HIV∆Env). Neutralization of mutant viruses was then assessed after treatment with PGT antibodies 121, 122, and 123 in order to determine which residues were critical in antibody recognition and binding. Here we show that none of the mutants produced led to a knock-out of neutralization with the exception of N332A, a mutation that removes the glycan at position 332. This glycan has already been found to be conformationally important for PGT121-123, even though these antibodies primarily bind peptide epitopes. These data suggest that the computational model will require additional refinement before it can be used as a predictive tool for antibody escape.
80