1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
3.3 Results
3.3.2 Phenotypic drug susceptibility
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171
0.0 0.2 0.4 0.6 0.8 1.0
0 2 4 6 8
p = 0.4206 r = 0.2024
IC50_LPV Viral load (log10 copies/ml)
0.0 0.2 0.4 0.6 0.8 1.0
0 2 4 6
8 p = 0.3760 r = 0.2220
IC50_DRV Viral load (log10 copies/ml)
0.0 0.2 0.4 0.6 0.8 1.0
0.0 0.5 1.0
1.5 p = 0.2293 r = -0.298
IC50_LPV
Replication capacity
0.0 0.2 0.4 0.6 0.8 1.0
0.0 0.5 1.0
1.5 p = 0.5153 r = -0.1641
IC50_DRV
Replication capacity
Figure 3-17 Correlation of IC50 with viral load and replication capacity for viruses treated with (a) lopinavir (LPV) or (b) darunavir (DRV). No correlation between IC50 and viral load or replication capacity was demonstrated for both LPV and DRV.
3.3.2.3 Number of mutations in Gag and Protease versus drug susceptibility
The relationship between number of mutations in Gag and Protease and drug susceptibility was assessed. The number of PR RAMs correlated positively with LPV and DRV IC50 (i.e. the higher the number of PR RAMs, the higher the IC50 for LPV and DRV) (Spearman’s correlation: rs=0.7404, p=0.0004, and rs=0.7255, p=0.0007, respectively) (Figure 3.18a). Conversely, the number of mutations in Gag did not correlate significantly with the IC50 of LPV or DRV (Figure 3.18b).
A
B
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0 2 4 6 8
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.0004 r = 0.7404
Number of PR resistance associated mutations
IC50_LPV
0 2 4 6 8
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.0007 r = 0.7255
Number of PR resistance associated mutations
IC50_DRV
0 1 2 3 4 5
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.6308 r = -0.1216
Number of gag resistance associated mutations
IC50_LPV
0 1 2 3 4 5
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.4047 r = 0.2092
Number of gag resistance associated mutations
IC50_DRV
Figure 3-18 Comparison between number of Protease (a) and Gag (b) resistance associated mutations and IC50 for lopinavir (LPV) and darunavir (DRV). (a) Spearman’s correlation showed that the number of PR RAMs correlated positively with IC50 for both LPV and DRV. (b) No significant correlation was noted between the number of Gag RAMs and IC50 for LPV or DRV.
3.3.2.4 Percent similarity to consensus vs drug susceptibility
The percent similarity of recombinant viruses to a consensus C sequence was computed.
The values generated (as described in section 3.3.1.6) were correlated with IC50 of LPV and DRV treated viruses in order to establish if sequence variation was associated with IC50.
There was a strong negative correlation between percent similarity of PR and IC50 for viruses treated with both LPV and DRV (Spearman’s correlation: rs=-0.7657, p=0.0009, and rs=-0.6762, p=0.0056 respectively) (Figure 3.19a). This showed, as expected, that viruses with similar PR sequences to the consensus C sequence had lower IC50 for both LPV and DRV. No significant relationship between sequence variation in Gag and IC50 for both LPV and DRV was observed (Figure 3.19b). This indicated that variations in Gag
A
B
173
were not significantly associated with altered IC50 for viruses treated with either LPV or DRV.
0.80 0.85 0.90 0.95 1.00
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.0009 r = -0.7657
% Similarity to consensus C Protease
IC50_LPV
0.80 0.85 0.90 0.95 1.00
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.0056 r = -0.6762
% Similarity to consensus C Protease
IC50_DRV
0.85 0.90 0.95 1.00
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.9805 r = 0.0061
% Similarity to consensus C Gag
IC50_LPV
0.85 0.90 0.95 1.00
0.0 0.2 0.4 0.6 0.8
1.0 p = 0.7353 r = -0.0857
% Similarity to consensus C Gag
IC50_DRV
Figure 3-19 Correlation between percent similarity of sequences for both Protease (a) and Gag (b) and IC50 of viruses treated with LPV or DRV. (a) There was a strong negative correlation between percent similarity of PR and IC50 for LPV and DRV. (b) No correlation between percent similarity of Gag and IC50 for viruses treated with LPV or DRV was shown.
3.3.2.5 Drug susceptibility of patient-derived Gag-Protease recombinant viruses
The 18 recombinant viruses assessed were stratified according to the presence of PR RAMs, rGag mutations (including 431V) and nGag mutations (including 69K). Viruses were divided into five groups for analysis as follows:
Four viruses with PR and rGag mutations (all viruses included harbored the 431V rGag mutation. Referred to as PR + rGag in Table 3.4);
Four viruses with PR RAMs, rGag mutations and nGag mutations (all viruses included harbored 69K. Referred to as PR RAMs + rGag + nGag in Table 3.4);
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B
174
Four viruses with rGag RAMs only (all viruses included harbored any rGag mutation, since 431V only occurred in the absence of PR RAMs in 1 virus.
Referred to as rGag in Table 3.4);
Three viruses with nGag mutations only (all viruses included harbored the 69K mutation. Referred to as nGag in Table 3.4) and
Three viruses with rGag and nGag mutations only (all viruses in this group harbored 69K. Referred to as rGag + nGag in Table 3.4).
Table 3.4 provides an overview of the 18 viruses used in the phenotypic drug susceptibility assay. Information is provided on the PR RAMs, rGag mutations and nGag mutations harboured by each virus. The FC in IC50 and associated standard deviation, for LPV and DRV, is provided for each virus. Additionally, the resistance profile/classification of each virus is provided, where S represents susceptible viruses and RS represents viruses with reduced susceptibility (cut-off values are described in section 3.2.3.3, Table 3.2).
Of the 18 viruses, ten (i.e. PCS002, PCSM002, PCS089, PCS134, PCS100, PCS071, PCS033, PCS153 and PCM029, PCS120) had reduced susceptibility to LPV and eight viruses (i.e. PCS011, PCS020, PCS049, PCS022, PCS096, PCS0128, PCS115 and PCS63) were susceptible to LPV (Table 3.4). Nine viruses that had reduced susceptibility to LPV also displayed reduced susceptibility to DRV, however the FC in IC50 was higher for LPV than DRV, suggesting that LPV IC50 is affected more so than DRV IC50 (Table 3.4). All eight viruses with PR RAMs displayed reduced susceptibility to LPV and DRV, indicating that viruses with PR RAMs require higher concentrations of PI’s to inhibit viral replication than that required by wild-type viruses.
Of the four viruses with rGag associated mutations only, one displayed reduced susceptibility to LPV and DRV (PCM029) whilst three were susceptible to LPV (PCS011, PCS020 and PCS049). Interestingly PCS049 harboured the 431V Gag resistance associated mutation which has been previously shown to confer resistance to all PI’s except DRV in the absence of PR RAMs. Whilst this virus was susceptible to DRV, it did not show a reduced susceptibility to LPV which was surprising (6, 7).
All three viruses harbouring the Q69K nGag amino acid substitution (PCS022, PCS096 and PCS128), were susceptible to LPV and DRV.