Demographic and clinical characteristics of study participants
Our study population consisted of 362 PLHIV, of whom 101 were ART-naïve, 118 on a regimen containing only RTIs and 143 on a regimen containing a PI/r. Females were more predominant (57.2%, 207/362), and the median [IQR] age was 41 [33-49] years. ART-naïve patients showed a significantly lower age compared to those treated (median [IQR], years: 38 [31-48] vs. 44 [32-52] in NNRTI-treated patients vs. 41 [33-47] in PI/r-treated patients; p=0.031). The overall median [IQR] CD4 cells count was 181 [60-361] cell/mm3, with no statistically significant difference observed among the three groups. Regarding viremia, ART-naïve patients had the highest median [IQR] viral load (5.6 [5.4-6.7] log10 copies/mL), followed by those on RTI-based regimens (5.3 [4.7-5.7] log10 copies/mL; versus on PI/r-based regimens (5.1 [4.3-5.6] log10 copies/mL; p<0.001). According to ART regimen, the majority of patients on RTI received TDF+3TC+EFV (52.5%), followed by AZT+3TC+NVP (19.5%); of the patients on PI/r, the majority received LPV/r or ATV/r containing regimen (65.7% and 32.9%, respectively) (Table 1).
Table 1
Socio-demographic characteristics of the study population
|
|
Naïve (n=101)
|
RTI-treated (n=118)
|
PI/r-treated (n=143)
|
p-valuea
|
Gender
|
Female n (%)
|
59 (28.5)
|
73 (35.3)
|
75 (36.2)
|
0.516
|
Male n (%)
|
42 (27.1)
|
45 (29.0)
|
68 (43.9)
|
Age (years), median [IQR]
|
38 [31-48]
|
44 [35-52]
|
41 [33-47]
|
0.031
|
CD4 (cells/µl), median [IQR]
|
231 [60-340]
|
169 [58-393]
|
167 [61-362]
|
0.760
|
Viral load (Log10/copies/mL), median [IQR]
|
5.61 [5.4-6.7]
|
5.3 [4.7-5.7]
|
5.1 [4.3-5.6]
|
<0.0001
|
ART regimen n (%)
|
2NRTIs+EFV
|
/
|
69 (58.4)
|
/
|
|
|
2NRTIs+NVP
|
/
|
49 (41.5)
|
/
|
|
2 NRTIs+LPV/r
|
/
|
/
|
94 (65.7)
|
|
2 NRTIs+ATV/r
|
/
|
/
|
47 (32.9)
|
|
2 NRTIs+IDV/r
|
/
|
/
|
2 (1.4)
|
|
Legend: IQR: Interquartile range; NRTIs: Nucleoside reverse transcriptase inhibitors; EFV: Efavirenz; NVP: Nevirapine; LPV/r: Lopinavir; ATV/r: Atazanavir; IDV/r: Indinavir; PI/r: ritonavir boosted protease inhibitors; RTI: Reverse transcriptase inhibitors. a: Chi-squared of independence test was used to estimate the potential differences. |
Viral subtypes distribution and protease drug resistance mutations profile among PI/r treated patients
Out of the overall 362 study participants, a broad diversity of HIV-1 non-B clades was found, driven by CRF02_AG (198, 54.69%), followed by pure subtypes A (49, 13.53%), D (23, 6.35%) and G (17, 4.69%). Among the 143 PI/r treated patients, the most represented subtype was also CRF02_AG subtype (87, 60.83%), follow by subtype A1 (22, 13.98%); G (12, 8.39%); CRF_11.cpx (9, 6.29%); F2 (6, 4.19%); D (2, 1.39%); C (2, 1.39%); CRF06_cpx (1, 0.69%), A2 (1, 0.69%); CRF01_AE (1, 0.69%) (Figure 1).
The overall prevalence of the presence of at least one protease drug resistance mutation among PI/r-experienced patients was 19.5% (n=28). The more frequent mutations were M46I (21; 14.69%), I84V (11, 7.69%) and I54V (11, 7.69%) (Figure 2).
P7 (NC)-P6 Gag mutations associated to PI/r exposure
By evaluating the last 56 amino acids of Gag sequences derived from 101 drug-naïve and 143 patients on PI/r containing regimen, we identified 18 mutations associated to PI/r exposure, based on the assumption that these mutations occurred with different frequencies in ART-naïve patients compared to patients on PI/r-based regimen.
These mutations were grouped into three classes, based on their prevalence in isolates from treatment naïve and PI/r treated individuals (Figure 3).
Class I included five mutations (L449F, D480N, L483Q, Y484P, T487V) that were completely absent or occurred with a frequency of <1% in isolates from drug-naïve patients and showed a significant increase in isolates from patients on PI/r-based regimen.
Class II included four mutations (S462L, I479G, I479K, D480E) already present in isolates from drug naïve patients at a frequency between 1% and 5% but with a significant increase in isolates from patients on PI/r-based regimen.
Class III included nine mutations (P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A) already present in isolates from drug naïve patients at a frequency ≥5% but with a significant increase in isolates from PI/r-based patients.
Gag P7(NC)-P6 mutations significantly associated to both reverse transcriptase inhibitors (RTI) and PI/r exposure
Four Gag P7(NC)-P6 mutations significantly associated to RTI exposure compared to naïve patients were also significantly associated to PI/r exposure (L449F, p=0.032; I479R, p=0.002; E482G, p=0.012; Y484P, p<0.0001) (Figure 3). Other Gag mutations P453L, E460A, S462L, R464G, S465F, V467E, Q474P, I479G, I479K, D480E, D480N, L483Q, T487A and T487V were only associated with PI/r exposure and not RTI when compared to ART naïve patients. Among the 18 Gag mutations significantly associated to PI/r exposure, ten mutations (P453L; E460A; R464G; V467E; I479K; Q474P; I479K; D480E; T487A; T487V) significantly differed between isolates from PI/r-treated vs RTI-treated (p<0.05, figure 3).
Gag P7(NC)-P6 mutations according to HIV-1 viral subtypes
Of the 18 mutations significantly associated to PI/r exposure, seven mutations were statistically different among HIV-1 subtypes. Indeed, in class 1, mutations L483Q, Y484P and T487V showed a significantly higher prevalence among subtype A1 infected individuals, when compared to other subtypes (p<0.001, Table 2). Of note, all the mutations with significantly varying frequencies of class 1 were found only in subtype A1 and other (mainly made of CRF11_cpx which has a portion of A subtype in Gag region). Regarding class 2, I479K was significantly more frequent in subtype categorized as other and D480E in subtype G, p<0.001. for class 3, S465F was significantly more frequent in subtype categorized as other and E482G in CRF02_AG, p<0.001.
Table 2
Gag P7(NC)-P6 mutations according to HIV-1 viral subtypes
Class
|
Mutations
|
Overall n (%)
|
Subtypes n (%)
|
CRF02_AG (n=87)
|
A1 (n=22)
|
G (n=12)
|
Others (n= 22)
|
p-valueb
|
|
L449F
|
8 (5.59)
|
3 (3.44)
|
1 (4.54)
|
1 (8.33)
|
3 (13.63)
|
0.305
|
|
D480N
|
8 (5.59)
|
3 (3.44)
|
2 (9.09)
|
0 (0.00)
|
3 (13.63)
|
0.450
|
I
|
L483Q
|
16 (11.18)
|
0 (0.00)
|
11 (50.00)
|
0 (0.00)
|
5 (22.72)
|
<0.001
|
|
Y484P
|
10 (6.99)
|
0 (0.00)
|
6 (27.27)
|
0 (0.00)
|
4 (18.18)
|
<0.001
|
|
T487V
|
14 (9.79)
|
0 (0.00)
|
11 (50.00)
|
0 (0.00)
|
3 (13.63)
|
<0.001
|
|
S462L
|
8 (5.59)
|
4 (4.59)
|
3 (13.63)
|
0 (0.00)
|
1 (4.54)
|
0.273
|
II
|
I479G
|
22 (15.38)
|
18 (20.68)
|
3 (13.63)
|
1 (8.33)
|
0(0.00)
|
0.161
|
|
I479K
|
54 (37.76)
|
25 (28.73)
|
7 (31.81)
|
4 (33.33)
|
18(81.81)
|
<0.001
|
|
D480E
|
36 (25.17)
|
16 (18.39)
|
1 (4.54)
|
9 (75.00)
|
10(45.45)
|
<0.001
|
|
P453L
|
22 (15.38)
|
10 (11.49)
|
6 (27.27)
|
5 (41.66)
|
1 (4.54)
|
0.190
|
|
E460A
|
142 (99.30)
|
86 (98.85)
|
22 (100.00)
|
12 (100.00)
|
22 (100.00)
|
1.000
|
|
R464G
|
138 (96.50)
|
87 (100.00)
|
18 (81.81)
|
11 (91.66)
|
22 (100.00)
|
0.403
|
III
|
S465F
|
24 (16.78)
|
0 (0.00)
|
0 (0.00)
|
8 (66.66)
|
16 (72.72)
|
<0.001
|
|
V467E
|
135 (94.40)
|
87 (94.25)
|
17 (77.27)
|
12 (66.66)
|
19 (86.36)
|
0.576
|
|
Q474P
|
99 (69.23)
|
57 (65.51)
|
16 (72.72)
|
4 (33.33)
|
22 (100.00)
|
0.320
|
|
I479R
|
63 (44.05)
|
39 (44.82)
|
10 (45.45)
|
5 (41.66)
|
9 (40.90)
|
0.162
|
|
E482G
|
74 (51.74)
|
68 (78.16)
|
2 (9.09)
|
2 (16.66)
|
2 (9.09)
|
<0.001
|
|
T487A
|
52 (36.36)
|
36 (41.37)
|
3 (13.63)
|
5 (41.66)
|
8 (36.36)
|
0.139
|
aeighteen (18) Gag mutations significantly associated to PI/r exposure; b Chi-squared of independence test was used to estimate the overall potential differences among types. The subtypes categorized as “other” included: CRF11_cpx (9), F2 (6), D (2), C (2), CRF06_cpx (1), A2 (1) and CRF01_AE (1). |
Covariation of Gag mutations with protease mutations
Another goal of our study was to assess the covariation of HIV Gag P7(NC)-P6 mutations with other mutations observed in the protease gene of 143 PI/r-treated patients, focusing our attention on PI/r major and/or accessory drug resistance mutations.
To identify significant patterns of pairwise correlations between Gag P7 (NC)-P6 mutations and protease mutations observed in isolates from PI/r-treated patients, we calculated the binomial correlation coefficient (phi) and its statistical significance for each pair of mutations (Table 2).
(i) Gag P7(NC)-P6 mutations involved in positive correlations with PI/r major resistance mutations
In Class I, two mutations were positively correlated (phi >0, P<0.001) as pairs with protease major resistance mutations: L449F correlated with the major resistance mutations V32I (phi= 0.38, p= 0.008), I54V (phi=0.27, p= 0.015) and L90M (phi= 0.32, p= 0.015); Y484P correlated with the major mutations I54V (phi= 0.33, p= 0.003) and V82A (phi= 0.26, p= 0.016). In Class II, D480E positively correlated with the major mutations I54V (phi= 0.31, p= P<0.001) and V82A (phi= 0.26, p=0.044). In Class III, only the Gag mutation P453L positively correlated with some major mutations as follows: L33F (phi= 0.28, p=0.004), M46I (phi= 0.30, p=0.001), I54L (phi= 0.27, p=0.022), I54V (phi= 0.38, p<0.001), V82A (phi= 0.20, p=0.031), V82T (phi= 0.27, p=0.021), and I84V (phi= 0.38, p<0.001), (Table 3).
Table 3
Significantly correlated pairs of HIV-1 Gag mutations with protease major or accessory resistance mutations.
Class
|
Gag Mutations
|
Frequency (%)a
|
Covariated Mutations
|
Frequency (%)b
|
Covariation Frequency (%)
|
phi
|
p-valued
|
|
L449F
|
8 (5.59)
|
L10F
|
6 (4.19)
|
2 (25.00)
|
0.25
|
3.69E-02
|
|
|
|
V32I
|
3 (2.09)
|
2 (25.00)
|
0.38
|
8.04E-03
|
|
|
|
M46I
|
22 (15.38)
|
4 (50.00)
|
-0.07
|
1.98E-02
|
I
|
|
|
I54V
|
11 (7.69)
|
3 (37.5)
|
0.27
|
1.55E-02
|
|
|
|
L90M
|
4 (2.79)
|
2 (25.00)
|
0.32
|
1.56E-02
|
|
Y484P
|
10 (6.99)
|
K20T
|
4 (2.79)
|
2 (20.00)
|
0.28
|
2.46E-02
|
|
|
|
L33F
|
9 (6.29)
|
3 (30.00)
|
0.26
|
1.68E-02
|
|
|
|
I54V
|
11 (7.69)
|
4 (40.00)
|
0.33
|
3.23E-03
|
|
|
|
V82A
|
9 (6.29)
|
3 (30.00)
|
0.26
|
1.68E-02
|
|
|
|
L89V
|
8 (5.59)
|
3 (30.00)
|
0.29
|
1.16E-02
|
|
I479G
|
22 (15.38)
|
I64M
|
19 (13.28)
|
7 (31.81)
|
-0.09
|
1.18E-02
|
II
|
D480E
|
36 (25.17)
|
V82A
|
9 (6.29)
|
5 (13.88)
|
0.18
|
4.47E-02
|
|
P453L
|
22 (15.38)
|
L10F
|
6 (4.19)
|
3 (13.63)
|
0.20
|
4.68E-02
|
|
|
|
L33F
|
9 (6.29)
|
5 (22.72)
|
0.28
|
4.62E-03
|
|
|
|
M46I
|
22 (15.38)
|
9 (40.90)
|
0.30
|
1.35E-03
|
|
|
|
I54L
|
2 (1.39)
|
2 (9.10)
|
0.27
|
2.28E-02
|
|
|
|
I54V
|
11 (7.69)
|
7 (31.81)
|
0.38
|
1.79E-04
|
III
|
|
|
Q58E
|
6 (4.19)
|
3 (13.63)
|
0.20
|
4.68E-02
|
|
|
|
V82A
|
9 (6.29)
|
4 (18.18)
|
0.20
|
3.19E-02
|
|
|
|
V82T
|
2 (1.39)
|
2 (9.10)
|
0.27
|
2.28E-02
|
|
|
|
I84V
|
11 (7.69)
|
7 (31.81)
|
0.38
|
1.79E-04
|
|
|
|
L89V
|
8 (5.59)
|
5 (22.72)
|
0.31
|
2.29E-03
|
|
S465F
|
24 (16.78)
|
I54V
|
11 (7.69)
|
5 (20.83)
|
0.22
|
2.03E-02
|
|
|
|
V82A
|
9 (6.29)
|
4 (16.67)
|
0.19
|
4.35E-02
|
|
Q474P
|
99 (69.23)
|
M46I
|
22 (15.38)
|
11 (11.11)
|
-0.03
|
4.46E-02
|
|
E482G
|
74 (51.74)
|
I54V
|
11 (7.69)
|
2 (2.70)
|
-0.19
|
2.71E-02
|
|
T487A
|
52 (36.36)
|
M46I
|
22 (15.38)
|
3 (5.76)
|
-0.21
|
1.64E-02
|
aThe frequency was determined in 143 isolates from PI/r-treated patients; bPercentages were calculated for patients containing each speciଁc mutation; cAll P values for covariation were signiଁcant at a false discovery rate of 0.05. Mutations in bold represents major protease resistance mutations. |
(i) Gag P7(NC)-P6 mutations involved in negative correlations with PI/r major resistance mutations
In Class I Gag mutations, L449F showed a significant negative correlation (phi<0 and P<0.05) with M46I (phi= -0.07, p=0.019); in Class II, I479G correlated with I64M (phi= -0.09, p=0.011); in class III, E482G correlated with I54V (phi= -0.19, p=0.027), Q474P with M46I (phi=-0.03, p=0.044) and T487A with M46I (phi= -0.21, p=0.016) (Table 3).
(ii) Gag P7(NC)-P6 mutations involved in correlations with PI/r non polymorphic accessory resistance mutations
Three Gag C-terminal mutations significantly associated with PI/r exposure showed significant positive correlations with specific PI/r non polymorphic accessory resistance mutations. In particular L449F correlated with L10F (phi=0.25, p= 0.036); P453L with L10F (phi=0.20, p= 0.046), Q58E (phi=0.20, p= 0.046) and L89V (phi=0.31, p= 0.002); Y484P with three PI/r accessory resistance mutations K20T (phi=0.28, p= 0.024), and L89V (phi=0.29, p= 0.011) (Table 3).
Clusters of correlated mutations
Because pairwise analysis suggested that Gag mutations are associated with speciଁc evolutionary pathways of known resistance-conferring mutations, we performed average linkage hierarchical agglomerative cluster analysis13 to investigate this hypothesis in more detail.
The dendrogram (Figure 4) shows that Gag mutation L449F and P453L significantly correlated to PI/r major resistance mutations. Specifically, P453L clustered (bootstrap value=0.33) with major PI/r resistance mutations M46I and I84V (covariation frequency:40.9% and 31.8%, respectively). Likewise, another cluster was formed by L449F and L90M (bootstrap value=0.75, covariation frequency: 25.0%).
Association of Gag P7(NC)-P6 mutations positively correlated to PI/r major resistance mutation with viral load and CD4 cell count at failure.
A further step in our study was to assess the difference in HIV viral load and CD4 cell count at the time of genotypic test between the patients harboring the Gag P7(NC)-P6 mutations positively correlated to PI/r major resistance mutations and those with wild type amino acids. Despite the slightly high median viremia in presence of two Gag mutations (L449F and Y484P) when compared to individuals with a wild-type residues, no significant variation was found in terms of viremia. Similarly, even though the median CD4 cells count was lower in all patients with Gag mutations when compared to wild type residues, no significant difference was found between the two groups in term of CD4 cell count (Table 4).
Table 4
Comparison of viremia and CD4 count of patients with wild type and mutated (positive correlated) amino acid
|
Frequency n (%) a
|
Viremia, median [IQR] Copies/ml b
|
Ratio
|
P-Valuec
|
CD4 count, median [IQR] cells/mm3b
|
Ratio
|
P-Valuec
|
Position
|
Residue
|
|
|
|
|
|
|
|
449
|
Ld
|
18 (12.68)
|
162116 [3725- 186450]
|
3.31
|
0.468
|
166 [46-443]
|
0.71
|
0.653
|
|
F
|
8 (5.59)
|
537857 [2689-720471]
|
118 [35-322]
|
484
|
Yd
|
23 (16.08)
|
90407 [4172-119019]
|
2.46
|
0.388
|
126 [74-467]
|
0.60
|
0.851
|
|
P
|
10 (6.99)
|
222565 [6093-298322]
|
107 [32-306]
|
480
|
Dd
|
88 (61.53)
|
255855 [3747-318872]
|
0.20
|
0.184
|
178 [78-489]
|
0.69
|
0.736
|
|
E
|
36 (25.17)
|
51807 [2226-74908]
|
124 [69-436]
|
453
|
Pd
|
116 (81.11)
|
172291 [6785-245815]
|
0.50
|
0.353
|
127 [81-351]
|
0.73
|
0.078
|
|
L
|
22 (15.38)
|
87814 [5535-98671]
|
93 [31-317]
|
a The frequency was determined in 143 PI-treated patients. bViremia and CD4 cell count values are contextual (±30 days) to the genotype resistance testing and were available for 110 PI/r treated patients (77% of samples). cP-values were determined by Mann-Whitney test. dThe wild-type amino acid in consensus B. |