Comparative Analysis of Drug Resistance Among B and the Most Prevalent Non-B HIV Type 1 Subtypes (C,F,and CRF02

Abstract

In recent years, increasing numbers of patients infected with HIV-1 non-B subtypes have been treated with modern antiretroviral regimens. Therefore, a better knowledge of HIV drug resistance in non-B strains is crucial. Thus, we compared the mutational pathways involved in drug resistance among the most common non-B subtypes in Italy (F, C, and CRF02_AG) and the B subtype. In total, 2234 pol sequences from 1231 virologically failing patients from Central Italy were analyzed. The prevalence of resistance mutations in protease and reverse transcriptase between non-B and B subtypes has been evaluated. Among patients treated with nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and with thymidine analogues (TA) experience, TAMs1 M41L and L210W were less prevalent in CRF02_AG, while TAMs2 T215F and K219E were more prevalent in the F subtype. In NRTI-treated patients having experience with abacavir, didanosine, tenofovir, or stavudine the K65R mutation was mostly prevalent in the C subtype. In non-NRTI (NNRTI)-treated patients infected by the C subtype the prevalence of K103N was lower than in patients infected with other subtypes, while the prevalence of Y181C and Y188L was higher compared to subtype B. The prevalence of Y181C was higher also in subtype F as compared to subtype B. In patients treated with protease inhibitors, L89V was predominantly found in CRF02_AG, while the TPV resistance mutation T74P was predominantly found in the C subtype. Some differences in the genotypic drug resistance have been found among patients infected with B, C, F, and CRF02_AG subtypes in relationship to treatment. These results may be useful for the therapeutic management of individuals infected with HIV-1 non-B strains.

Introduction

T he human immunodeficiency virus type 1 (HIV-1) evolves rapidly due to high replication rates, changing selective pressures, and the error-prone reverse transcriptase (RT).

To date, in the viral M group nine subtypes (A, B, C, D, F, G, H, J, and K) have been described, together with many recombinant forms called, respectively, circulating recombinant form (CRF) and unique recombinant form (URF).^1,2 HIV-1 subtype B is the predominant variant in North America, the Caribbean, Latin America, Western and Central Europe, and Australia, while most HIV-1 infections worldwide are caused by subtype A (East Africa), subtype C (East and Southern Africa, Ethiopia, and India), CRF02_AG (West Africa), or CRF01_AE (Asia).^3
–5

However, several studies showed that non-B subtype HIV-1 infections have been rapidly increasing during the past years in previously subtype B homogeneous areas such as Europe and North America.^6

–9 For example, in France and in Switzerland it is estimated that non-B infections constitute roughly 20% and 35% of HIV-1 infections,^6,8 with an increased prevalence of CRFs over the past 5 years. A similar situation has been observed in Italy, where HIV-1 non-B-infected patients increased from 0.3% in 1993 to 20% in recent years.⁹ Several studies performed in this country have showed that the most prevalent non-B subtypes in this country are C, F, and the recombinant form CRF02_AG.^10

–16

In this regard, Italy is a good example of the way the spread of non-B subtypes is highly dependent upon several variables such as the demographics of local HIV-1 epidemics and their evolution over time: while the prevalence of non-B HIV-1 subtypes has remained low in South Italy, such as Sicily,¹⁵ it has increased in North Italy, with a preponderance of C and F subtypes due to the increased number of immigrants from Africa and Eastern Europe.¹⁴

Although non-B infections are infrequent in North America, a study in New York identified non-B infections in a few U.S. citizens who never traveled abroad, suggesting that transmission of non-B subtype occurs in the United States independently of travel history.⁷

Due to the spread of non-B viruses and the introduction of antiretroviral drugs in developing countries (known for the largest assortment of non-B subtypes), further knowledge concerning the responsiveness to antiretroviral therapy and HIV-1 drug resistance in non-B strains is required. In this regard, it is known that different viral subtypes can be naturally less or more susceptible to specific drugs.^17,18 For example, the recombinant form CRF02_AG is more susceptible to nelfinavir (NFV) and ritonavir (RTV) than subtypes C and F, subtype G is more sensitive to tipranavir (TPV) and lopinavir (LPV) than other subtypes,¹⁹ and subtype C has accelerated risk in developing resistance to tenofovir (TDF).^20
–22 An explanation for the extreme variability of HIV-1 subtypes in the highly active antiretroviral therapy (HAART) response can be given by the presence of some polymorphisms, which can influence both the emergence of drug resistance mutations as well as the response to drugs. For example, polymorphisms at residues 20 and 36 of HIV-1 protease decrease the genetic barrier to TPV resistance in subtypes A, C, F, and G,² while nucleotide heterogeneity at 64 and 65 RT positions accelerated development of K65R in subtype C.^20,23

However, comparative data about the pathways of resistance of these subtypes versus the B subtype are still incomplete in the European settings, where patients carrying B and non-B subtypes are treated with similar antiviral regimens.

Moreover, data about these issues are sometimes insufficient to set and validate drug resistance interpretation algorithms in populations infected with non-B variants.

Thus, the goal of this study was to compare the mutational pathways involved in resistance to nucleoside/nucleotide RT inhibitors (NRTIs), non-NRTI (NNRTIs), and protease inhibitors (PIs) among HIV-1 B and non-B (with particular attention to subtypes F and C and the recombinant form CRF02_AG) subtype-infected patients, with similar drug experiences, followed in clinical centers in Central Italy over the years 2001–2008.

Materials and Methods

Study population

The study included a total of 2234 pol sequences from 1231 virologically failing patients followed in several clinical centers in Rome and surroundings, undergoing a genotyping resistance test (GRT) over the years 2001–2008. Among these 1231 patients, 189 were infected by non-B strains. The most frequent HIV-1 non-B subtypes in our cohort were subtypes F (47, 24.9%), C (45, 23.8%), and the recombinant form CRF02_AG (36, 19.1%). At the time of GRT, all patients analyzed were under treatment and had a plasma viral load >50 copies/ml. Data for all patients were stored in a specifically designed anonymous database that included genotypic, demographic, immunologic, virologic, and therapeutic parameters.

HIV sequencing

HIV genotype analysis was performed on plasma samples by means of a commercially available kit (ViroSeq HIV-1 genotyping system; Abbott Laboratories). Briefly, RNA was extracted, retrotranscribed by murine leukemia virus RT, and amplified with Amplitaq-Gold polymerase enzyme by using two different sequence-specific primers for 40 cycles. Pol-amplified products (containing the entire PR and the first 335 amino acids of the RT open reading frame) were sequenced full-length in sense and antisense orientations by an automated sequencer (ABI 3100) by using seven different overlapping sequence-specific primers.²⁴ Sequences having a mixture of wild-type and mutant amino acid residues at single positions were considered to have the mutant(s) at that position.

Phylogenetic analysis

All HIV-1 pol sequences (1302 nucleotides) were aligned and compared with reference sequences for the major HIV-1 subtypes, available at http://hiv-web.lanl.gov/content/hivdb/SUBTYPE_REF/align.html using CLUSTAL X.²⁵ The sequences were then manually edited with the Bioedit program,²⁶ and gaps were removed from the final alignment. All sequences were analyzed using the REGA HIV-1 subtyping tool.²⁷ Separate trees were then generated using F84 model of substitution with both neighbor-joining (NJ) and maximum likelihood (ML) tree building methods,²⁸ for both non-B pure subtypes and putative recombinant forms. Phylogenetic trees were performed with different evolutionary model according to the Hierarchical Likelihood Ratio Test (HLRT) implemented in the Model Test V3.0 software.²⁹ The statistical robustness within each phylogenetic tree was confirmed with a bootstrap analysis using 1000 replicates for the NJ tree. All calculations were performed with PAUP*4.0 software.²⁸

Mutation prevalence

The prevalence of resistance mutations in the three most prevalent non-B subtypes (C, F, and CRF02_AG) and the 1042 B subtype-infected patients has been evaluated. For the analysis of drug resistance mutations, to avoid any relationship between the mutation prevalence in the different subtypes and drug pressure, the HIV-1-infected patients were divided into six different subgroups according to duration and type of antiretroviral treatment: (1) patients treated with thymidine analogues (TAs) zidovudine (AZT) and/or stavudine (d4T) for the analysis of the prevalence of TA-related mutations (TAMs: M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q); (2) patients treated with experience to d4T for the analysis of K65R^30
–32; (3) patients treated with abacavir (ABC), didanosine (ddI), and/or tenofovir (TDF) for the analysis of the prevalence of ABC/ddI/TDF-related mutations K65R, D67G, T69ins, K70E/G, and L74V^30,31; (4) patients treated with lamivudine (3TC) and/or emtricitabine (FTC) for the analysis of M184V^30,31; (5) patients treated with NNRTI for the analysis of the prevalence of NNRTI-related mutations (L100I, K101E/P, K103N/S, V106A/M, V108I, V179F, Y181C/I/V, Y188C/H/L, G190A/E/S, P225H, M230L, and K238T)^30,31; and (6) patients treated with PI for the analysis of the prevalence of PI-related mutations (D30N, V32I, L33F, M46I/L, I47A/V, G48V, I50L/V, I54L/M, Q58E, T74P, L76V, V82A/F/L/S/T, I84A/C/V, N83D, N88D/S, L89V, and L90M).^30,31,33,34 For each subgroup one sequence per patient (the last one available under treatment) was analyzed.

Statistical analysis

To correctly compare rates of mutation prevalence among populations of different size (HIV-1 subtype B-infected patients vs. HIV-1 subtype non-B C, F, or CFR02_AG-infected patients), 50 sequences from the original HIV-1 subtype B-infected population were drawn randomly 500 times. The mutation prevalence found in each randomized HIV-1 B sample was then compared with that found in the subtypes C, F and CRF02_AG by Fisher exact test. p values<0.05 were considered statistically significant for all tests utilized. Random selection of 500 samples was obtained by coding a specific Java program. Moreover, statistically significant differences in the prevalence of each drug resistance mutation and in the duration of therapy among the three most prevalent non-B subtype groups were assessed by a 3×2 Chi squared test and Kruskal–Wallis test, respectively. Again, p values<0.05 were considered statistically significant. The calculations of all statistical test were performed by the R open source software.

Results

Patients characteristics

Demographic and therapeutic characteristics of HIV-1-infected patients stratified by HIV-1 subtype are reported in Table 1. At GRT, viral load, and CD4 cell count were comparable among patients infected by different HIV-1 subtypes (p>0.05). Analyzing the antiretroviral treatment, patients infected by CRF02_AG had a median duration of therapy of 168 (interquartile range, IQR: 71–285) weeks, significantly lower than that observed for patients infected by C [188 (111–382) weeks], F [368 (226–551) weeks], or B subtype [415 (234–614) weeks] (p=0.03).

Table 1.

Patients' Characteristics

	Subtypes
Characteristic	Overall non-B	C	F	AG	B
Patients, N	189	45	47	36	1042
Male, N (%)	107 (58.4)	18 (42.8)	35 (74.5)	16 (45.7)	756 (72.8)
Italian, N (%)	31 (30.0)	4 (14.3)	16 (64.0)	3 (13.6)	634 (89.0)
HIV exposure, N (%)
MSM	18 (13.1)	2 (6.5)	8 (22.2)	2 (7.7)	144 (18.1)
Heterosexual	107 (78.1)	29 (93.5)	23 (63.8)	23 (88.5)	271 (34.0)
IDU	6 (4.4)	0 (0.0)	2 (5.5)	1 (3.8)	357 (44.9)
Other	6 (4.4)	0 (0.0)	3 (8.3)	0 (0.0)	24 (3.0)
Age, years, median (IQR)	38.1 (32.7–45.3)	37.3 (32.5–42.2)	40.0 (35.4–46.6)	32.8 (29.1–39.7)	42.4 (38.1–46.9)
Viremia at GRT (log copies/ml), median (IQR)	3.3 (2.6–4.3)	3.2 (2.5–4.3)	3.4 (2.4–4.5)	3.2 (2.8–4.5)	3.8 (2.9–4.5)
CD4 cell count at GRT (cells/μl), median (IQR)	286 (152–476)	303 (154–420)	248 (114–483)	342 (247–509)	307 (160–506)
Year of ART initiation, N (%)
≤2000	79 (42.0)	19 (42.2)	26 (55.3)	9 (25.8)	716 (69.4)
2001–2003	51 (27.1)	10 (22.2)	13 (27.7)	13 (37.1)	195 (18.9)
≥2004	58 (30.9)	16 (35.6)	8 (17.0)	13 (37.1)	121 (11.7)
Patients failing first line regimen, N (%)	28 (14.8)	8 (17.8)	5 (10.6)	15 (41.7)	168 (16.1)
Number of ART regimens, median (IQR)	3 (2–5)	2 (1–5)	4 (2–6)	2 (1–3)	3 (2–6)
ART duration,^a weeks, median (IQR)	237 (85–418)	188 (111–382)	368 (226–551)	168 (71–285)	415 (234–614)

ART duration indicates the time elapsed between the start of the first line regimen and the genotypic resistance test.

Prevalence was calculated on the samples with available data (range: 80% of all samples).

GRT, genotypic resistance test; AG, CRF02_AG; ART, antiretroviral therapy; IDU, injection drug users; MSM, males who have sex with males; IQR, interquartile range; N, number of patients.

Information about current and previous antiretrovirals used is shown both in Table 1 and Supplementary Table S1. (Supplementary Data are available online at www.liebertonline.com/aid.)

Mutation prevalence

NRTI resistance mutations

Among patients experienced with TA, the percentage of patients infected by CRF02_AG strains with TA resistance was quite less (4, 14.3%) than that observed for patients infected by C and F subtypes [9 (25.0%), 14 (34.1%), p=0.178]. In particular, TAMs1 M41L and L210W were less prevalent in patients with CRF02_AG compared to patients with F and C subtypes (Table 2); this difference was larger and statistically significant when compared with the B subtype (p=0.001 and p=0.005, respectively). Differently, TAMs2 T215F and K219E were more prevalent in patients infected with the F subtype compared with those infected with the C, CRF02_AG (completely absent in this viral strain), and B subtype (p=0.015 and p=0.03, respectively).

Table 2.

Prevalence of Drug Resistance Mutations in Patients Failing Highly Active Antiretroviral Therapy According to HIV-1 Subtype and Treatment

						p value ^b (% of significance)
Mutation	C	F	AG	p value ^a	B	C vs. B	F vs. B	AG vs. B
NRTI-treated pts with TA exp, N	36	41	28		984
M41L	8 (22.2)	10 (23.4)	1 (3.6)		299 (29.1)			0.001 (97.8)
D67N	5 (13.9)	8 (19.5)	3 (10.7)		198 (20.1)
K70R	2 (5.5)	6 (14.6)	3 (10.7)		122 (12.4)
L210W	3 (8.3)	5 (12.2)	0 (0.0)		185 (18.8)			0.005 (89.0)
T215Y	6 (16.7)	6 (14.6)	4 (14.3)		277 (28.2)
T215F	1 (2.8)	6 (14.6)	0 (0.0)	0.03	46 (4.7)		0.015 (32.2)
K219Q	2 (5.5)	1 (2.4)	1 (3.6)		49 (5.0)
K219E	0 (0.0)	5 (12.2)	0 (0.0)	0.02	41 (4.2)		0.03 (11.2)
NRTI-treated pts with d4T exp, N	25	25	11		717
K65R	6 (24.0)	1 (4.0)	0 (0.0)		23 (3.2)	<0.001 (60.8)
NRTI-treated pts with ddI or TDF or ABC exp, N	35	41	18		734
K65R	6 (17.1)	1 (2.4)	0 (0.0)	0.02	28 (3.8)	0.003 (42.0)
D67G	0 (0.0)	1 (2.4)	0 (0.0)		0 (0.00)
T69ins	1 (2.9)	1 (2.4)	0 (0.0)		1 (0.1)
K70E	1 (2.8)	0 (0.0)	1 (5.6)		4 (0.5)
K70G	0 (0.0)	0 (0.0)	0 (0.0)	NC	5 (0.7)
L74V	1 (2.8)	7 (17.1)	0 (0.0)	0.03	69 (9.4)
NRTI-treated pts with 3TC/FTC exp, N	43	44	31		1009
M184V	19 (44.2)	22 (50.0)	14 (45.2)		545 (54.0)
NNRTI-treated pts, N	20	23	10		335
L100I	0 (0.0)	2 (8.7)	0 (0.0)		17 (5.1)
K101E	2 (10)	0 (0.0)	0 (0.0)		30 (8.9)
K101P	1 (5)	0 (0.0)	0 (0.0)		7 (2.09)
K103N	5 (25)	14 (60.9)	4 (40)	0.06	168 (50.2)	0.04 (35.0)
K103S	2 (10)	0 (0.0)	0 (0.0)		7 (2.1)
V106A	0 (0.0)	0 (0.0)	0 (0.0)	NC	18 (5.4)
V106M	1 (5)	0 (0.0)	0 (0.0)		3 (0.9)
V108I	4 (20)	5 (21.7)	0 (0.0)		45 (13.4)
Y181C	8 (40)	9 (39.1)	2 (20)		66 (19.7)	0.04 (22.2)	0.03 (46.6)
Y181I	1 (5.0)	0 (0.0)	0 (0.0)		1 (0.1)
Y188C	0 (0.0)	0 (0.0)	0 (0.0)	NC	1 (0.1)
Y188L	3 (15)	2 (8.7)	1 (10.0)		9 (2.7)	0.02 (22.8)
G190A	5 (25)	1 (4.3)	1 (10.0)		61 (18.2)
G190E	0 (0.0)	0 (0.0)	0 (0.0)	NC	5 (1.5)
G190S	1 (5)	1 (4.2)	1 (10)		15 (4.5)
P225H	2 (10)	4 (16.7)	0 (0.0)		32 (9.6)
M230L	0 (0.0)	0 (0.0)	0 (0.0)	NC	9 (2.7)
K238T	0 (0.0)	2 (8.7)	0 (0.0)		28 (1.4)
PI-treated pts, N	32	39	27		612
D30N	0 (0)	0 (0)	0 (0)	NC	49 (8.0)
V32I	1 (3.1)	3 (7.7)	0 (0.0)		30 (4.9)
L33F	1 (3.1)	4 (10.3)	0 (0)		53 (8.7)
M46I	6 (18.8)	6 (15.4)	1 (3.7)		114 (18.6)
M46L	1 (3.1)	4 (10.3)	2 (7.4)		54 (8.8)
I47A	1 (3.1)	2 (5.1)	0 (0)		1 (0.2)
I47V	1 (3.1)	1 (2.6)	0 (0)		27 (4.4)
G48V	0 (0)	0 (0)	0 (0)	NC	17 (2.8)
I50L	0 (0)	1 (2.6)	0 (0)	NC	4 (0.6)
I50V	1 (3.1)	1 (2.6)	0 (0)		8 (1.3)
I54L	0 (0)	0 (0)	0 (0)	NC	15 (2.4)
Q58E	1 (3.1)	3 (7.7)	0 (0)		26 (4.2)
T74P	4 (12.5)	2 (5.1)	0 (0)		7 (1.1)	0.001 (51.0)
L76V	0 (0)	2 (5.1)	1 (3.7)		10 (1.6)
V82A	3 (9.4)	8 (20.5)	1 (3.7)		91 (14.9)
V82F	0 (0.0)	0 (0.0)	0 (0.0)	NC	6 (1.0)
V82L	0 (0.0)	0 (0.0)	0 (0.0)	NC	3 (0.5)
V82S	1 (3.1)	3 (7.7)	0 (0.0)		30 (4.9)
V82T	0 (0)	0 (0)	0 (0)	NC	20 (3.3)
I84A	0 (0)	0 (0)	0 (0)	NC	0 (0.0)
I84C	0 (0)	0 (0)	0 (0)	NC	0 (0.0)
I84V	1 (3.1)	3 (7.7)	0 (0)		55 (9.0)
N88D	0 (0)	0 (0)	0 (0)	NC	36 (5.9)
N88S	0 (0)	1 (2.6)	2 (7.4)		6 (1.0)
L89V	3 (9.4)	1 (2.6)	4 (14.8)		13 (2.1)			0.004 (29.4)
L90M	8 (25)	9 (23.1)	4 (14.8)		150 (24.5)

Differences in the prevalence of each resistance mutation among the three non-B subtypes were assessed by the Chi square test (2×3 table). Only significant p values (p < 0.05) are reported.

Differences in the prevalence of each drug resistance mutation between non-B subtypes and B subtype were assessed by the Fisher exact test. This test was repeated for each one of the 500 B subtype resamplings. Only the percentage of significant p values (p < 0.05) among the 500 resamplings are reported. The NNRTI mutations V179F, Y181V, and Y188H and the protease mutations I54M and N83D were completely absent in patients analyzed in the present study.

ABC, abacavir; AG, CRF02_AG; 3TC, lamivudine; ddI, didanosine; d4T, stavudine; Exp, experience; FTC, emtricitabine; 3TC, lamivudine; N, number of patients; NNRTI, nonnucleoside reverse transcriptase inhibitors; NRTI, nucleoside reverse transcriptase inhibitors; PI, protease inhibitors; Pts, patients; TA, thymidine analogs; TDF, tenofovir; NC, not calculable.

As previously reported, the prevalence of K65R was known to be significantly higher in patients infected with the C subtype and with experience with ABC, ddI, or TDF than those infected with F, CRF02_AG, and B subtypes.^20,21 Our results confirmed these data, but highlighted a higher prevalence of K65R in patients infected with subtype C and with experience with d4T (p<0.001). In particular, as shown in Table 3, experience with d4T, as well as experience with ddI or TDF or ABC, influenced the presence of K65R at GRT only in subtype C-infected patients (p=0.01).

Table 3.

Prevalence of the K65R Mutation in Patients Failing Highly Active Antiretroviral Therapy According to HIV-1 Subtype and Treatment

						p value ^b
NRTI-treated patients at GRT with	C	AG	F	p value ^a	B	C vs. B	AG vs. B	F vs. B
Exp to ddI or TDF or ABC or d4T	21	18	26		405
Therapy duration,^c weeks, median (IQR)	68 (33–100)	47 (33–74)	67 (26–127)		68 (40–104)
K65R prevalence, N (%)	4 (19.0)	0 (0.0)	0 (0.0)	0.03	17 (4.2)	0.01
Exp to ddI or TDF or ABC, but no exp to d4T	17	8	22		141
Therapy duration,^c weeks, median (IQR)	57 (17–109)	37 (23–51)	27 (24–92)		49 (26–78)
K65R prevalence, N (%)	1 (5.9)	0 (0.0)	0 (0.0)		6 (4.2)
Exp to d4T, but no exp to ddI or TDF or ABC	2	3	3		28
Therapy duration,^c weeks, median (IQR)	72 (44–100)	42 (29–54)	77 (54–104)		73 (37–109)
K65R prevalence, N (%)	1 (50.0)	0 (0.0)	0 (0.0)		1 (3.6)

Differences in the prevalence of K65R among the three non-B subtypes were assessed by the Chi square test (2×3 table).

Differences in the prevalence of K65R between non-B subtypes and the B subtype were assessed by the Fisher exact test.

Therapy duration indicates the total weeks under ddI, TDF, d4T, or ABC treatment.

The prevalence of the K65R mutation was calculated in groups of patients treated with NRTI and with a similar median therapy duration at the moment of the genotypic resistance test. Only significant p values (p<0.05) are reported in the table.

ABC, abacavir; AG, CRF02_AG; d4T, stavudine; ddI, didanosine; Exp, experience; GRT, genotypic resistance test; NRTI, nucleoside reverse transcriptase inhibitors; TDF, tenofovir.

NNRTI resistance mutations

In NNRTI-treated patients, the mutation K103N was present at lower frequency in subtype C compared with F, CRF02_AG, and with the B subtype (p=0.04) (Table 2). This finding was confirmed also after stratifying the population for nevirapine (NVP) or efavirenz (EFV) experience (data not shown). Conversely, the mutations Y181C and Y188L were found with higher frequency in patients infected with subtype C compared to patients infected with subtype B (p=0.04 and p=0.02). The Y181C was also found with a higher frequency in subtype F when compared with subtype B (p=0.03). As expected, this mutation was more prevalent in patients with NVP experience than in patients treated with EFV [NVP experience: C (66.7%), F (87.5%) vs. B (31.2%): p=0.07 and p=0.002, respectively; EFV experience: C (25.0%), F (26.3%) vs. B (8.6%): p<0.001 and p=0.03, respectively].

PI resistance mutations

In PI-treated patients, L90M was the most common mutation found in each subtype group (Table 2). The mutation D30N was completely absent in patients infected with CRF02_AG, subtype C, and subtype F while its prevalence reached 8.0% in patients infected with subtype B; this result was also confirmed when the analysis was performed only in NFV experienced patients (0% in non-B subtypes vs. 15.0% in B subtypes, p=0.02, data not shown). Interestingly, the mutation L89V (potentially associated with resistance to fosamprenavir and, to a lesser extent, to darunavir and lopinavir) was predominantly found in CRF02_AG (p=0.004), while the TPV resistance mutation T74P was predominantly found in subtype C (p=0.001), even if only one patient was treated with TPV.

Discussion

The present study provides new information about resistance profiles occurring in patients infected with several HIV-1 non-B subtypes (C, F, and CFR02_AG) and the B subtype.

An improved knowledge of the significance of non-B subtypes for resistance evolution and interpretation is becoming mandatory today not only because antiretroviral therapy is being introduced in countries where non-B subtypes are driving the epidemic, but also because the number of infections by these variants is increasing sharply in several European countries.^6,8,9

The results obtained in this study show that HIV-1 drug resistance does not emerge at the same rate among different HIV-1 subtypes, and that different pathways to resistance are taken in different proportions according to subtype under the same therapeutic pressure. For example, differences in the prevalence of some TAMs1 (M41L and L210W, but not T215Y) were observed among NRTI-treated patients with TA experience infected by B and C, F, and CRF02_AG strains. In particular in patients infected with the recombinant form CRF02_AG, the prevalence of M41L was lower than that observed for other subtypes, and L210W was completely absent. In this regard, the absence of this latter mutation in patients infected with CRF02_AG and its lower prevalence in patients infected with subtypes C and F in comparison to those infected with subtype B could be due to the increased genetic barrier of these subtypes toward the development of an L210W substitution. CRF02_AG, C, and F viral strains frequently contain either the CTG, CTA, or TTA codon at this position (all encoding for leucine), instead of TTG, the most common codon found in the B subtype (also encoding for leucine).³⁵ So, while in subtype B the L210W substitution required only one transversion (from TTG to TGG, total score=2.5), in subtype non-B, because of the presence of other starting codons, this substitution required one transition and one transversion (total score=1+2.5=3.5). Thus, this observation can suggest that the genetic barrier, based upon the different codon heterogeneities naturally present in different subtypes, may play a relevant role in the selection of mutations at failure.

It is important to note that even if in our cohort of patients, those infected by subtype C and CRF02_AG had a median duration of treatment lower than patients infected by subtypes F and B. The low prevalence of TAMs1 in CRF02_AG was also confirmed by analyzing a subgroup of NRTI-treated patients with TA experience and with similar therapy duration (data not shown). This finding was also confirmed in our resistance database including more than 8000 HAART-failing patients: patients infected by CRF02_AG failed their HAART regimen with fewer M41L and L210W mutations than patients infected with other HIV-1 subtypes (4.8% in CRF02_AG vs. 7.9% in C subtype vs. 20% in F subtype vs. 21% in B subtype, p<0.001; M.M. Santoro, unpublished data). Other studies confirmed the lower prevalence of TAMs in the CRF02_AG-infected population when compared with B or other non-B subtypes.^18,36

Similarly, the TAMs2 T215F and K219E were completely absent in the CRF02_AG population; however, these mutations were generally rare also in subtype B-infected and subtype C-infected patients (<5%). Their prevalence reached 14% and 12% only in subtype F-infected patients. These patients also showed a high prevalence of other TAMs (in particular M41L, D67N, and K70R), suggesting an overall more rapid selection of TAMs during a TA regimen in patients infected with subtype F compared with other subtypes. Interestingly, the high prevalence of mutations at RT positions 67 and 70 in F subtypes was also recently found in a cohort of Brazilian HAART-treated patients, where these mutations reached 30%.³⁷ The molecular reasons for this selection in subtype F are still unknown, and it does not seem to be associated with a different genetic barrier at that position.³⁵

Our study confirmed the finding that the K65R mutation in the RT occurs more frequently in patients infected with subtype C than in patients infected by other HIV-1 subtypes.³⁸ However, we found a high prevalence of this mutation in subtype C-infected patients exposed not only to TDF but also to an ABC, ddI, and d4T-containing regimen. This result suggests a role for K65R not only as a TDF resistance mutation,³⁹ but also as ABC, ddI, and d4T selected mutation, especially in patients infected with the C subtype.^32,40,41

Regarding NNRTI mutations, our study showed that different resistant mutational pathways might characterize virologic failure in patients infected with subtypes B, C, F, and CRF02_AG. In particular, in patients infected with subtype C, a lower frequency of the K103N mutation and a higher prevalence of Y188L and Y181C than in patients infected with subtype B have been observed. While subtype C showed a low prevalence of K103N, as many as 60.9% of subtype F carried this mutation. Interestingly, we also found a higher prevalence of the NRTI mutation L74V in subtype F than in subtype C and CRF02_AG. Moreover, four out of the seven HIV-1 subtype F-infected patients with L74V also carried K103N. The association of these two mutations can be explained by the compensatory role of L74V, known to restore the replication capacity impaired by the selection of K103N.^42,43

Different results were obtained in the several studies comparing the prevalence of NNRTI-related mutations in different HIV-1 non-B subtypes. For example, the K103N mutation was relatively more frequent in subtype C-infected women failing NNRTI-based therapy than subtype A and D in a Uganda study.⁴⁴ The mutations G190A/S were seen as frequent polymorphisms in subtype C Israelian-infected patients, but not in Indian C-infected patients.^45,46 Finally, many studies confirmed that the V106M mutation is frequently seen in non-B subtypes (especially C and CRF02_AE) after therapy with EFV or NVP.^45

–48 A higher prevalence of G190A/S and V106M was also observed in the present study in patients infected by the C subtype in comparison to other ones, even if these differences were not statistically significant. Overall, these differences may have clinical relevance, in view of the increasing use of second generation NNRTIs, such as etravirine or rilpivirine, whose efficacy is affected differently by the various mutations selected by first generation NNRTIs. In particular, K103N has no effect upon etravirine efficacy, while others, such as Y181C, Y188L, and G190A/S, are more relevant. Additional new studies will be necessary to better define the prevalence of these mutations, in view of the appropriate use of etravirine or rilpivirine in NNRTI-failing patients carrying non-B subtypes.

Regarding PI mutations, no significant differences in the prevalence of major resistance mutations were observed in patients infected with B, C, F, or CRF02_AG subtypes, with the exception of T74P and L89V. In particular, the TPV resistance mutation T74P was more frequently found in patients infected with subtype C, even if only one patient had experience with TPV, while mutation L89V was more frequently identified in patients infected with CRF02_AG. Of interest, the NFV mutation D30N was completely absent in non-B subtypes, even if around >35% of patients experienced this drug in the previous or current regimen, suggesting that this mutation is rarely selected in subtypes other than B.⁴⁹ All together, these findings may suggest that the different prevalence of drug resistance mutations in the different subtypes can be due to more intrinsic properties of the virus and not only to different pressures of antiretrovirals. For mutations T74P and L89V, their different prevalence is also supported by a different genetic barrier observed in different subtypes. Indeed, by analyzing 2357 HIV-1 pol sequences obtained from naive HIV-1-infected patients, more than 10.0% of drug-naive patients infected by HIV-1 subtype C showed the TCA codon (encoding serine) instead of the codon ACA (encoding threonine) at position 74. This serine codon displays a lower genetic barrier for the development of the resistance substitution proline (encoded by CCA, one transition, score=1) compared to the threonine codon (encoded by ACA, one transversion, score=2.5).

In the case of protease position 89, the wild-type codon in most of the non-B subtypes is the ATG (M), itself conferring resistance in most of the non-B subtypes,^33,50 in contrast to subtype B sequences, which show 89M in only 4.8% of cases, preferring the CTG (L) (present in 86.2% of subtype B). Genetic barrier analysis showed that the ATG codon has a lower resistance score to evolve into the resistance substitution GTG (V) (one transition, score=1) compared to codon CTG (L) (one transversion, score=2.5).

Interestingly, the CRF02_AG recombinant form was the non-B subtype with the highest frequency of codon ATG (M) at position 89 (95.4% of patients), if compared with F and C subtypes (F=73.0% and C=81.9%, respectively). Of note, these two protease positions 74 and 89 seem to be closely related to each other. As shown by Gonzales et al., the protease mutation L89I/V is stabilized by the acquisition of T74S in subtype G.⁵¹ It is conceivable that in subtype C, the mutation T74S, a frequent PR polymorphism in drug-naive patients, could favor the 89I/V selection under treatment. These findings suggest that some natural polymorphisms (such as T74S and L89M) should be considered with particular attention in the therapeutic management of patients infected by HIV-1 non-B subtypes. It should be remembered that they could favor the emergence of resistance mutations. For instance, it has been proposed that the presence of methionine at protease position 89 in non-B subtype viruses favors the selection of mutation I/V at position 89 and the PI major mutation L90M, rather than the lysine in subtype B.³³

In conclusion, although the present exploratory analysis was limited to a small number of patients for each subtype and requires confirmation by further studies, these findings suggest that the genetic diversity within HIV-1 group M could play a role in the development of resistance to antiviral drugs. A tendency to a lower level of resistance to AZT was observed in patients infected with CRF02_AG HIV-1 variants, while a higher level of resistance to TDF and TAs was observed for subtype C and F, respectively. The therapeutic implications of these unique subtype differences in terms of long-term efficacy of different antiviral regimens are still unknown. They will be best explored in ad hoc studies, to be done in the future.

Footnotes

Acknowledgments

This work was financially supported by grants from the Italian National Institute of Health, the Italian Ministry of Instruction University & Research (MIUR), Current and Finalized Research of the Italian Ministry of Health, and the European Commission Framework 7 Programme (CHAIN, the Collaborative HIV and Anti-HIV Drug Resistance Network, Integrated Project no. 223131), and AVIRALIA Foundation. We would like to thank Daniele Armenia, Mario Santoro, Andrea Biddittu, Domenico Di Pinto, Marzia Romani, Francesca Stazi, Fabio Continenza, Daniele Pizzi, AlbertoGiannetti, Anna Pacifici, and Massimo Giuliani for sequencing and data management.

Maria Mercedes Santoro and Claudia Alteri contributed equally to this work.

This work was in part presented at the 7th and 8th European HIV Drug Resistance workshop (7th: Stockholm, Sweden, March 25–27, 2009, Abstract 130; 8th: Sorrento, Italy, March 15–17, 2010, Abstract 87).

Author Disclosure Statement

No competing financial interests exist.

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Comparative Analysis of Drug Resistance Among B and the Most Prevalent Non-B HIV Type 1 Subtypes (C,F,and CRF02_AG) in Italy

Abstract

Introduction

Materials and Methods

Study population

HIV sequencing

Phylogenetic analysis

Mutation prevalence

Statistical analysis

Results

Patients characteristics

Mutation prevalence

NRTI resistance mutations

NNRTI resistance mutations

PI resistance mutations

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material